Metal-glycoprotein complexes and photodynamic therapy of immune privileged sites with same

ABSTRACT

Compositions of the invention include glycoproteins, such as transferrin, and metal-based coordination complexes, which are preferably chemotherapeutic compounds and more preferably tunable photodynamic compounds. The compositions are useful as in vivo diagnostic agents, and as therapeutic agents for treating or preventing diseases including those that involve hyperproliferating cells in their etiology, such as cancer. Compositions of the invention are further capable of destroying microbial cells, such as bacteria, fungi, and protozoa, and destroying viruses. Treatment methods of the invention can treat conditions throughout the body, including conditions located across the blood-brain barrier, the retina-blood barrier and the blood-cerebrospinal fluid barrier.

BACKGROUND OF THE INVENTION

1. Field of Invention

This invention relates to metal-based coordination complexes, and moreparticularly to photodynamic therapy using metal-glycoprotein complexesas photo dynamic compounds.

2. Description of Related Art

Photodynamic therapy (PDT) is currently an active area of research forthe treatment of diseases associated with hyperproliferating cells suchas cancer and non-malignant lesions. The development of new photodynamiccompounds (PDCs or photosensitizers, PSs) for photodynamic therapy (PDT)has been increasingly focused on metallosupramolecular complexes derivedfrom metals. For example, WO 2013158550 A1 and WO 2014145428 A2 disclosemetal-based PDCs useful as in vivo diagnostic agents, as therapeuticagents for treating or preventing diseases that involve unwanted and/orhyperproliferating cell etiology, including cancer, as agents fortreating infectious diseases, and as agents for pathogen disinfectionand/or sterilization. U.S. Pat. No. 6,962,910, U.S. Pat. No. 7,612,057,U.S. Pat. No. 8,445,475 and U.S. Pat. No. 8,148,360 disclosesupramolecular metal complexes capable of cleaving DNA when irradiatedlow energy visible light with or without molecular oxygen.

Delivery of metal-based coordination complexes and PDCs to biologicaltargets can pose a challenge, which many have attempted to address.

For example, US 20120264802 discloses photosensitizer compounds based onfunctionalized fullerenes useful in targeted PDT, and methods of usethereof.

WO 2013020204 A1 discloses biodegradable polymeric nanoparticlescomprising an inner core formed of a photodynamic agent capable of beingactivated to generate cytotoxic singlet oxygen. These nanoparticles haveanti-cell proliferation activity and are useful in treating bothcancerous and non-cancerous conditions including actinic keratosis,psoriasis and acne vulgaris. Preferably, the photodynamic agent is ahypocrellin B derivative while the polymeric nanoparticle comprisespolyglycolic acid, polylactic acid or poly(lactide-co-glycolide).Hypocrellin-comprising nanoparticles are demonstrated to be activated bylight or hydrogen peroxide.

US20110288023 discloses modified Transferrin (Tf) molecules andconjugates of the Tf molecules with a therapeutic agent. Also disclosedare methods of treating cancer wherein the therapeutic agents arechemotherapeutic agents. The modified Tf molecules improve the deliveryof the conjugated therapeutic agent to a target tissue.

WO 2002094271 A1 discloses a homogeneous conjugate for targeting andtreating diseased cells wherein the conjugate comprises an anti-cancerdrug and a targeting protein, wherein said anti-cancer drug is selectedfrom the group consisting of heat sensitizers, photosensitizers andapoptosis inducing compounds, a method for making such a conjugate, andmethods for using the conjugate. The targeting protein is preferablytransferrin.

U.S. Pat. No. 7,001,991 discloses a homogeneous conjugate for targetingand treating diseased cells wherein the conjugate comprises ananti-cancer drug and a targeting protein, wherein said anti-cancer drugis selected from the group consisting of heat sensitizers,photosensitizers and apoptosis inducing compounds, a method for makingsuch a conjugate, and methods for using the conjugate. The targetingprotein is preferably transferrin.

U.S. Pat. No. 7,809,428 discloses PDT methods for treatment ofvulnerable plaques by selectively targeting and/or eliminating theinflammatory components of vulnerable plaques. In a preferredembodiment, photosensitizer compositions are coupled to macromolecularcarriers that target T cells of vulnerable plaques. These macromolecularcarriers can be targeted to, for example, IL-10, receptor, monocyteinflammatory protein-1 and receptors thereof and transferrin. Suchmacromolecular carriers can be, for example, antibodies against thesebiomolecules, ligands binding the same or analogs thereof, including,but not limited to monoclonal antibodies that recognize CD1, CD2, CD3,CD4, CDS, CD6, CD7, CDB, CD25, CD28, CD44, CD71 or transferrin.

Large (>500 Da) PSs are difficult to apply topically. Non-selectivity ofdelivery is another problem. Various patch- and film-based topicalapplication formulations and methods of enhanced delivery of PSsdirectly into cancer cells have been proposed to overcome bothdifficulty of delivery of large (>500 Da) PS molecules andnon-selectivity of the delivery. They include various patch- andfilm-based topical application formulations (Donnelly et al 2009), redoxactivation (Graf, Lippard, 2012), receptor-mediated delivery (Nkepang etal., 2014), photoinduced delivery (Chen et al., 2014; Yin et al., 2014;Sardar et al., 2014), liposomes (Temizel et al., 2014; Muehlmann et al.,2011), and delivery using nanoparticles including fullerenes (Biju,2014; Yuan, Liu, 2014; Zhen et al., 2014; Wong et al., 2013; Yang etal., 2014; He et al., 2014). Combining of transferrin with fullerenes isalso proposed (Zhang et al., 2015) as well as conjugation of PS-loadedliposomes with many molecules (folate, growth factors, glycoproteinssuch as transferrin, glycolipids) receptors for which are upregulated incancer cells (Muehlmann et al., 2011; Nkepang et al., 2014). PEGylatedAIPcS4-loaded liposomes conjugated with transferrin were used againstcervical cancer cells (Gijsens et al., 2002). Exploration of Tfconjugation on the efficiency of liposome-encapsulated PS Foscan, achlorine-based photosensitizer, in PDT of esophageal cancer was,however, not successful, likely due to the destabilization of theliposomes (Paszko et al., 2013).

Protein-based delivery systems include systems based on albumin(nanoparticles system), small heat shock protein, viral capsid andapoferritin (protein cage systems) used for doxorubicin delivery, soyprotein (film-based system) for methylene blue delivery (MaHam et al.,2009). Apoferritin (i.e. Ferritin that is not combined with iron, aprotein of 450 kDa) was used to encapsulate various cytostaticanticancer drugs: doxorubicin, carboplatin, cisplatin, daunorubicinalthough immune response to apoferritin may be a drawback (Heger et al.,2014). Among PCs, encapsulation of Methylene blue into apo-ferritinallowed increasing singlet oxygen production and enhancement ofcytotoxic effects on cells (Heger et al., 2014, review).

The use of transferrin with liposomes containing aluminum phthalocyaninetetrasulfonate (LiposomesAlPcS₄) is disclosed by Derycke et al., 2014;Gaspar et al., 2012.

None of the foregoing references explicitly propose the use oftransferrin in combination with metal-based photosensitizers. Such usewas disclosed by the inventor in US 20160206653 A1.

Despite the foregoing developments, it is still desired to provideimproved compositions and methods for delivering PDCs to biologicaltargets. It is further desired to provide increased efficacy ofselective uptake of PDCs by biological targets. It is further desired toimprove intracellular uptake of Ruthenium, Ruthenium-Rhodium andOsmium-based photosensitizers predominantly by cancer cells and tumortissues. It is further desired to increase PDC efficacy at longerwavelengths. It is further desired to improve absorbance, ROS productionand PDT effect of the Ruthenium, Ruthenium-Rhodium and Osmium-basedphotosensitizers. It is further desired to improve the PDT effect inhypoxia. It is further desired to improve targeting of immune privilegedsites.

BRIEF SUMMARY OF THE INVENTION

A first aspect of the invention comprises a composition comprising:

a metal-binding glycoprotein; and

a chemotherapeutic compound containing at least one transition metalpreferably selected from the group consisting of Ru, Rh and Os,

wherein the composition has at least one of the following enhancedproperties relative to the chemotherapeutic compound without theglycoprotein: (a) increased uptake by cancer cells; (b) increased uptakeby tumors; (c) increased efficacy at wavelengths longer than 600 nm; (d)increased efficacy at wavelengths less than or equal to 600 nm; (e)improved absorbance at wavelengths longer than 600 nm; (f) improvedabsorbance at wavelengths less than or equal to 600 nm; (g) increasedproduction of reactive oxygen species; (h) increased photodynamictherapy effect under non-hypoxic conditions; (i) increased photodynamictherapy effect under hypoxic conditions; (j) increased LD50; (k)increased MTD; (l) increased photostability; and (m) increasedshelf-life.

In certain embodiments, the glycoprotein is transferrin and thechemotherapeutic compound has the formula (I):

including hydrates, solvates, pharmaceutically acceptable salts,prodrugs and complexes thereof, wherein:M at each occurrence is independently a transition metal, which ispreferably selected from the group consisting of osmium, ruthenium andrhodium;X is selected from the group consisting of Cl⁻, PF₆ ⁻, Br⁻, BF₄ ⁻, ClO₄⁻, CF₃SO₃ ⁻, and SO₄ ⁻²;n=0, 1, 2, 3, 4, or 5;q is independently at each occurrence 0, 1, or 2;y is independently at each occurrence 0, 1, or 2;z is independently at each occurrence 1, 2, or 3;Lig¹ is a bidentate ligand that at each occurrence is each independentlyselected from the group consisting of

Lig² is a bidentate ligand that at each occurrence is each independentlyselected from the group consisting of

Lig³ is a bidentate ligand that at each occurrence is each independentlyselected from the group consisting of

R¹ is selected from the group consisting of hydrogen, optionallysubstituted phenyl, optionally substituted aryl, optionally substitutedheteroaryl, 4-pyridyl, 3-pyridyl, 2-thiazole, 2-pyrolyl, 2-furanyl,

R^(2a), R^(2b), R^(2c), R^(2d), R^(2e), R^(2f), R^(2g), R^(2h), R^(2i),R^(2j), R^(2k), and R^(2l) at each occurrence are each independentlyselected from the group consisting of hydrogen, C₁₋₆ optionallysubstituted alkyl, C₁₋₆ optionally substituted branched alkyl, C₃₋₇optionally substituted cycloalkyl, C₁₋₆ optionally substitutedhaloalkyl, C₁₋₆ optionally substituted alkoxy, CO₂R⁵, CONR⁶ ₂, NR⁷ ₂,SO₃H, sulfate, sulfonate, optionally substituted aryl, optionallysubstituted aryloxy, optionally substituted heteroaryl, and optionallysubstituted heterocycle;R^(3a), R^(3b), R^(3c), R^(3d), R^(3e), R^(3f), R^(3g), R^(3h), R^(3i),R^(3j), R^(3k), and R^(3l) at each occurrence are each independentlyselected from the group consisting of hydrogen, C₁₋₆ optionallysubstituted alkyl, C₁₋₆ optionally substituted branched alkyl, C₁₋₆optionally substituted haloalkyl, C₁₋₆ optionally substituted alkoxy,optionally substituted phenyl, and CO₂R⁸;R^(4a), R^(4b), and R^(4c) at each occurrence are each independentlyselected from the group consisting of hydrogen, C₁₋₆ optionallysubstituted alkyl, C₁₋₆ optionally substituted branched alkyl, C₁₋₆optionally substituted cycloalkyl, C₁₋₆ optionally substitutedhaloalkyl, C₁₋₆ optionally substituted alkoxy, CO₂R⁵, CONR⁶ ₂, NR⁷ ₂,sulfate, sulfonate, optionally substituted aryl, optionally substitutedaryloxy, optionally substituted heteroaryl, and optionally substitutedheterocycle;R^(4a) and R^(4b) at each occurrence on a thiophene ring are takentogether with the atom to which they are bound to form an optionallysubstituted ring having from 6 ring atoms containing 2 oxygen atoms;R⁵ at each occurrence are each independently selected from the groupconsisting of hydrogen and optionally substituted alkyl;R⁶ at each occurrence are each independently selected from the groupconsisting of hydrogen and optionally substituted alkyl;R⁷ at each occurrence are each independently selected from the groupconsisting of hydrogen and optionally substituted alkyl; andR⁸ at each occurrence are each independently selected from the groupconsisting of hydrogen and optionally substituted alkyl.

In certain embodiments, the glycoprotein is transferrin and thechemotherapeutic compound has the formula (VI):

including hydrates, solvates, pharmaceutically acceptable salts,prodrugs and complexes thereof wherein;M¹ and M² at each occurrence is independently a transition metal, and ispreferably independently selected from the group consisting of osmium,manganese, molybdenum, rhenium, ruthenium, iron, cobalt, rhodium,iridium, nickel, platinum, and copper;A² is selected from the group consisting of

t is an integer.

In certain embodiments, the glycoprotein is transferrin and thechemotherapeutic compound has the formula (VIIa)

including hydrates, solvates, pharmaceutically acceptable salts,prodrugs and complexes thereof wherein:M¹ and M² at each occurrence is independently a transition metal, and ispreferably independently selected from the group consisting of osmium,manganese, molybdenum, rhenium, ruthenium, iron, cobalt, rhodium,iridium, nickel, platinum, and copper;Lig¹ is a bidentate ligand that at each occurrence is each independentlyselected from the group defined above;Lig³ is a bidentate ligand that at each occurrence is each independentlyselected from the group defined above;p is independently at each occurrence 0, 1, or 2;q is independently at each occurrence 0, 1, or 2;n is 0, 1, 2, 3, 4, or 5; andA³ is selected from the group consisting of

In certain embodiments, the glycoprotein is transferrin and thechemotherapeutic compound has the formula (II)

-   -   including hydrates, solvates, pharmaceutically acceptable salts,        prodrugs and complexes thereof, wherein:    -   M is a transition metal preferably selected from the group        consisting of manganese, molybdenum, rhenium, iron, ruthenium,        osmium, cobalt, rhodium, iridium, nickel, platinum, and copper;    -   X is selected from the group consisting of Cl⁻, PF₆ ⁻, Br⁻, BF₄        ⁻, ClO₄ ⁻, CF₃SO₃ ⁻, and SO₄ ⁻²;    -   n=0, 1, 2, 3, 4, or 5;    -   y=1, 2, or 3;    -   z=0, 1, or 2;    -   Lig at each occurrence is independently selected from the group        consisting of

R¹ is selected from the group consisting of

-   u is an integer;-   R^(2a), R^(2b), R^(2c), R^(2d), R^(2e), and R^(2f) at each    occurrence are each independently selected from the group consisting    of hydrogen, C1-6 optionally substituted alkyl, C1-6 optionally    substituted branched alkyl, C3-7 optionally substituted cycloalkyl,    C1-6 optionally substituted haloalkyl, C1-6 optionally substituted    alkoxy, CO₂R⁵, CONR⁶ ₂, NR⁷ ₂, sulfate, sulfonate, optionally    substituted aryl, optionally substituted aryloxy, optionally    substituted heteroaryl, and optionally substituted heterocycle;-   R^(3a), R^(3b), R^(3c), R^(3d), R^(3e), R^(3f), R^(3g), R^(3h)    R^(3i), R^(3j), R^(3k), R^(3l), and R^(3m) at each occurrence are    each independently selected from the group consisting of hydrogen,    C1-6 optionally substituted alkyl, C1-6 optionally substituted    branched alkyl, C1-6 optionally substituted haloalkyl, C1-6    optionally substituted alkoxy, and CO₂R⁸;-   R^(4a), R^(4b), and R^(4c) at each occurrence are each independently    selected from the group consisting of hydrogen, C1-6 optionally    substituted alkyl, C1-6 optionally substituted branched alkyl, C1-6    optionally substituted cycloalkyl, C1-6 optionally substituted    haloalkyl, C1-6 optionally substituted alkoxy, CO₂R⁵, CONR⁶ ₂, NR⁷    ₂, sulfate, sulfonate, optionally substituted aryl, optionally    substituted aryloxy, optionally substituted heteroaryl, and    optionally substituted heterocycle;-   R^(4a) and R^(4b) at each occurrence on a thiophene ring are taken    together with the atom to which they are bound to form an optionally    substituted ring having from 6 ring atoms containing 2 oxygen atoms;-   R⁵ at each occurrence is independently selected from the group    consisting of hydrogen and optionally substituted alkyl;-   R⁶ at each occurrence is independently selected from the group    consisting of hydrogen and optionally substituted alkyl;-   R⁷ at each occurrence is independently selected from the group    consisting of hydrogen and optionally substituted alkyl; and-   R⁸ at each occurrence is independently selected from the group    consisting of hydrogen and optionally substituted alkyl.

A second aspect of the invention comprises a method for treating acondition associated with hyperproliferating cells, said methodcomprising administering to a subject having the condition a metalbinding glycoprotein and a chemotherapeutic compound containing at leastone transition metal, wherein the metal binding glycoprotein and thechemotherapeutic compound containing at least one transition metal areadministered in a combined amount effective to treat the condition.

In certain embodiments of the treatment method, the subject isirradiated with light effective to activate the chemotherapeuticcompound.

In certain embodiments of the treatment method, the metal-bindingglycoprotein is effective to provide at least one of the followingadvantages relative to treatment by the chemotherapeutic compoundwithout the glycoprotein: (a) increased uptake by cancer cells; (b)increased uptake by tumors; (c) increased efficacy at wavelengths longerthan 600 nm; (d) increased efficacy at wavelengths less than or equal to600 nm; (e) improved absorbance at wavelengths longer than 600 nm; (f)improved absorbance at wavelengths less than or equal to 600 nm; (g)increased production of reactive oxygen species; (h) increasedphotodynamic therapy effect under non-hypoxic conditions; (i) increasedphotodynamic therapy effect under hypoxic conditions; (j) increasedLD50; (k) increased MTD; (l) increased photostability; and (m) increasedshelf-life.

In certain embodiments of the treatment method, the metal-bindingglycoprotein and the chemotherapeutic compound containing at least onetransition metal are administered in a combination dosage form or areco-administered from two or more independent sources.

In certain embodiments of the treatment method, the metal-bindingglycoprotein is transferrin and the chemotherapeutic compound is definedby formula (I), formula (VI), formula (VIIa) or formula (II) above.

In certain embodiments of the treatment method, the at least onetransition metal is at least one of Ru, Rh, Os and Ir.

In certain embodiments of the treatment method, the metal-bindingglycoprotein is a recombinant human transferrin, such as OPTIFERRIN.

In certain embodiments of the treatment method, the condition treated bythe method is at an immune privileged site, the metal bindingglycoprotein and the chemotherapeutic compound containing at least onetransition metal are systemically administered in a form of a complex,and the complex crosses at least one of the blood-brain barrier, theretina blood barrier and the blood-cerebrospinal fluid barrier toaccumulate at the immune privileged site. Preferably, the metal-bindingglycoprotein is transferrin and the chemotherapeutic compound is definedby formula (I), formula (VI), formula (VIIa) or formula (II) above.

In certain embodiments of the treatment method, the complex crosses theblood-brain barrier and preferentially accumulates in a brain tumorbeing treated by the method.

In certain embodiments, the subject is irradiated with ionizingradiation effective to activate the chemotherapeutic compound.

In certain embodiments of the treatment method, the metal bindingglycoprotein and the chemotherapeutic compound containing at least onetransition metal are administered in a form of a complex, thechemotherapeutic compound is a photodynamic compound, and the complexfurther comprises at least one active pharmaceutical ingredient (API).In some of these embodiments, the API is an anti-neoplastic agent freeof any transitional metals and/or the complex crosses at least one ofthe blood-brain barrier, the retina blood barrier and theblood-cerebrospinal fluid barrier to accumulate at an immune privilegedsite.

A third aspect of the invention comprises a method for destroying amicrobial cell, said method comprising: contacting the microbial cellwith an effective amount of the composition according to the invention;and irradiating the microbial cell with light effective to activate thecomposition so as to destroy the microbial cell.

These and other objects, features, and advantages will become apparentto those of ordinary skill in the art from a reading of the followingdetailed description and the appended claims. All percentages, ratiosand proportions herein are by weight, unless otherwise specified. Alltemperatures are in degrees Celsius (° C.) unless otherwise specified.All documents cited are in relevant part, incorporated herein byreference; the citation of any document is not to be construed as anadmission that it is prior art with respect to the present invention.

BRIEF DESCRIPTION OF SEVERAL VIEWS OF THE DRAWINGS

The invention will be described in conjunction with the followingdrawings in which like reference numerals designate like elements andwherein:

FIGS. 1A, 1B and 1C show graphs of absorbance/absorbance increaseagainst wavelength.

FIGS. 2A, 2B and 2C show graphs of absorbance against wavelength.

FIGS. 3A and 3B show graphs of fluorescence against wavelength.

FIGS. 4A and 4B show graphs of fluorescence against wavelength.

FIGS. 5A and 5B show graphs of fluorescence against wavelength.

FIGS. 6A, 6B and 6C show graphs of absorbance ratio against incidentphotons.

FIGS. 7A, 7B and 7C show graphs of absorbance ratio against incidentphotons.

FIGS. 8A, 8B and 8C show graphs of absorbance ratio against incidentphotons.

FIGS. 9A, 9B and 9C show graphs of absorbance ratio against incidentphotons.

FIGS. 10A, 10B and 10C show graphs of cellular uptake of metals bycontrol and test samples.

FIGS. 11A, 11B and 11C show graphs of cellular uptake of metals bycontrol and test samples.

FIGS. 12A and 12B show graphs of cell kill with and without transferrin.

FIGS. 13A and 13B show graphs of cell kill with and without transferrin.

FIGS. 14A and 14B show graphs of cell kill with and without transferrin.

FIGS. 15A and 15B show graphs of cell kill with and without transferrin.

FIGS. 16A, 16B and 16C show graphs of cell kill with and withouttransferrin.

FIGS. 17A, 17B, 17C and 17D show graphs of cell kill with and withouttransferrin.

FIGS. 18A, 18B, 18C and 18D show graphs of cell kill with and withouttransferrin.

FIGS. 19A, 19B, 19C and 19D show graphs of cell kill with and withouttransferrin.

FIGS. 20A and 20B show graphs of cell kill with and without transferrinand with and without antibodies.

FIGS. 21A and 21B show graphs of cell kill with and without transferrinand with and without antibodies.

FIGS. 22A and 22B show graphs of cell kill with and without transferrinand with and without antibodies.

FIGS. 23A and 23B show graphs of uptake of metals over time by muscleand tumor tissue with and without transferrin.

FIGS. 24A and 24B show graphs of uptake of metals over time by muscleand tumor tissue with and without transferrin.

FIGS. 25A and 25B show graphs of uptake of metals over time by muscleand tumor tissue with and without transferrin.

FIG. 26 shows a graph of percent survival against days elapsed afterPDT.

FIG. 27 shows a graph of percent survival against days elapsed afterPDT.

FIG. 28 shows a graph of molar extinction coefficient againstwavelength.

FIG. 29 shows a graph of molar extinction coefficient againstwavelength.

FIG. 30 shows a graph of optical density ratio against incident photonnumber.

FIG. 31 shows graphs of optical density against wavelength.

FIG. 32 shows graphs of molar extinction coefficient ratio againstenergy.

FIG. 33 shows graphs of optical density against wavelength.

FIG. 34 shows graphs of optical density against wavelength.

FIGS. 35A, 35B, 35C, 35D, 35E, 35F, 35G and 35H are Magnetic ResonanceImages (MRIs) of the brain of a rat treated in accordance with anembodiment of the invention.

FIGS. 36A, 36B, 36C, 36D, 36E, 36F, 36G, 36H, 36I and 36J are MRIs ofthe brain of another rat treated in accordance with another embodimentof the invention.

FIGS. 37, 38 and 39 show inductively coupled plasma mass spectrometry(ICP-MS) data.

FIG. 40 shows a graph of in vitro cell kill vs. concentration.

FIG. 41A is a bar chart showing in vitro cell kill by TLD1433 with andwithout transferrin, wherein the TLD1433 was not photodynamicallyactivated.

FIG. 41B is a bar chart showing in vitro cell kill by TLD1433 with andwithout transferrin, wherein the TLD1433 was photodynamically activated.

FIG. 41C is a bar chart showing in vitro cell kill by TLD1433 with andwithout OPTIFERRIN, wherein the TLD1433 was not photodynamicallyactivated.

FIG. 41D is a bar chart showing in vitro cell kill by TLD1433 with andwithout OPTIFERRIN, wherein the TLD1433 was photodynamically activated.

FIG. 42A is a bar chart showing in vitro cell kill by TLDOSH2DPPN withand without transferrin, wherein the TLDOSH2DPPN was notphotodynamically activated.

FIG. 42B is a bar chart showing in vitro cell kill by TLDOSH2DPPN withand without transferrin, wherein the TLDOSH2DPPN was photodynamicallyactivated.

FIG. 42C is a bar chart showing in vitro cell kill by TLDOSH2DPPN withand without OPTIFERRIN, wherein the TLDOSH2DPPN was not photodynamicallyactivated.

FIG. 42D is a bar chart showing in vitro cell kill by TLDOSH2DPPN withand without OPTIFERRIN, wherein the TLDOSH2DPPN was photodynamicallyactivated.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS OF THE INVENTION Glossary

Throughout the description, where compositions are described as having,including, or comprising specific components, or where processes aredescribed as having, including, or comprising specific process steps, itis contemplated that compositions of the present teachings also consistessentially of, or consist of, the recited components, and that theprocesses of the present teachings also consist essentially of, orconsist of, the recited processing steps.

In the application, where an element or component is said to be includedin and/or selected from a list of recited elements or components, itshould be understood that the element or component can be any one of therecited elements or components and can be selected from a groupconsisting of two or more of the recited elements or components.

The use of the singular herein includes the plural (and vice versa)unless specifically stated otherwise. In addition, where the use of theterm “about” is before a quantitative value, the present teachings alsoinclude the specific quantitative value itself, unless specificallystated otherwise.

It should be understood that the order of steps or order for performingcertain actions is immaterial so long as the present teachings remainoperable. Moreover, two or more steps or actions can be conductedsimultaneously

For the purposes of the present invention the terms “compound,”“analog,” and “composition of matter” stand equally well for theinventive compounds described herein, be they photodynamic or not,including all enantiomeric forms, diastereomeric forms, salts, and thelike, and the terms “compound,” “analog,” and “composition of matter”are used interchangeably throughout the present specification.

Compounds described herein can contain an asymmetric atom (also referredas a chiral center), and some of the compounds can contain one or moreasymmetric atoms or centers, which can thus give rise to optical isomers(enantiomers) and diastereomers. The present teachings and compoundsdisclosed herein include such enantiomers and diastereomers, as well asthe racemic and resolved, enantiomerically pure R and S stereoisomers,as well as other mixtures of the R and S stereoisomers andpharmaceutically acceptable salts thereof. Optical isomers can beobtained in pure form by standard procedures known to those skilled inthe art, which include, but are not limited to, diastereomeric saltformation, kinetic resolution, and asymmetric synthesis. The presentteachings also encompass cis and trans isomers of compounds containingalkenyl moieties (e.g., alkenes and imines). It is also understood thatthe present teachings encompass all possible regioisomers, and mixturesthereof, which can be obtained in pure form by standard separationprocedures known to those skilled in the art, and include, but are notlimited to, column chromatography, thin-layer chromatography, andhigh-performance liquid chromatography.

Pharmaceutically acceptable salts of compounds of the present teachings,which can have an acidic moiety, can be formed using organic andinorganic bases. Both mono and polyanionic salts are contemplated,depending on the number of acidic hydrogens available for deprotonation.Suitable salts formed with bases include metal salts, such as alkalimetal or alkaline earth metal salts, for example sodium, potassium, ormagnesium salts; ammonia salts and organic amine salts, such as thoseformed with morpholine, thiomorpholine, piperidine, pyrrolidine, amono-, di- or tri-lower alkylamine (e.g., ethyl-tert-butyl-, diethyl-,diisopropyl-, triethyl-, tributyl- or dimethylpropylamine), or a mono-,di-, or trihydroxy lower alkylamine (e.g., mono-, di- ortriethanolamine). Specific non-limiting examples of inorganic basesinclude NaHCO₃, Na₂CO₃, KHCO₃, K₂CO₃, Cs₂CO₃, LiOH, NaOH, KOH, NaH₂PO₄,Na₂HPO₄, and Na₃PO₄. Internal salts also can be formed. Similarly, whena compound disclosed herein contains a basic moiety, salts can be formedusing organic and inorganic acids. For example, salts can be formed fromthe following acids: acetic, propionic, lactic, benzenesulfonic,benzoic, camphorsulfonic, citric, tartaric, succinic, dichloroacetic,ethenesulfonic, formic, fumaric, gluconic, glutamic, hippuric,hydrobromic, hydrochloric, isethionic, lactic, maleic, malic, malonic,mandelic, methanesulfonic, mucic, naphthalenesulfonic, nitric, oxalic,pamoic, pantothenic, phosphoric, phthalic, propionic, succinic,sulfuric, tartaric, toluenesulfonic, and camphorsulfonic as well asother known pharmaceutically acceptable acids.

When any variable occurs more than one time in any constituent or in anyformula, its definition in each occurrence is independent of itsdefinition at every other occurrence (e.g., in N(R⁶)₂, each R⁶ may bethe same or different than the other). Combinations of substituentsand/or variables are permissible only if such combinations result instable compounds.

The terms “treat” and “treating” and “treatment” as used herein, referto partially or completely alleviating, inhibiting, ameliorating and/orrelieving a condition from which a patient is suspected to suffer.

As used herein, “therapeutically effective” and “effective dose” referto a substance or an amount that elicits a desirable biological activityor effect.

As used herein, the term “photodynamic therapy” refers to a treatmentfor destroying cells or modulating immune function, including immuneresponse, of cells and tissue through use of a drug that can beactivated by light of a certain wavelength and dose.

As used herein the term “chemotherapeutic compound” refers to a chemicalcompound with prophylactic, ameliorative and/or curative properties withrespect to one or more conditions or diseases.

As used herein, the term “photodynamic compound” refers to a compoundthat provides photodynamic therapy. Photodynamic compounds are a subsetof chemotherapeutic compounds as defined herein.

Except when noted, the terms “subject” or “patient” are usedinterchangeably and refer to mammals such as human patients andnon-human primates, as well as experimental animals such as rabbits,rats, and mice, and other animals. Accordingly, the term “subject” or“patient” as used herein means any mammalian patient or subject to whichthe compounds of the invention can be administered. In an exemplaryembodiment of the present invention, to identify subject patients fortreatment according to the methods of the invention, accepted screeningmethods are employed to determine risk factors associated with atargeted or suspected disease or condition or to determine the status ofan existing disease or condition in a subject. These screening methodsinclude, for example, conventional work-ups to determine risk factorsthat may be associated with the targeted or suspected disease orcondition. These and other routine methods allow the clinician to selectpatients in need of therapy using the methods and compounds of thepresent invention.

As used herein, the term “biological target” refers to an organ, tissueand/or cell of an organism and/or to the organism itself.

Advantages of the Invention

The invention is based in part on the unexpected discovery that Ru, Rhand Os based PDCs, particularly those disclosed in WO 2013158550 A1, WO2014145428 A2, U.S. Pat. No. 6,962,910, U.S. Pat. No. 7,612,057, U.S.Pat. No. 8,445,475, U.S. Pat. No. 8,148,360 and US 20160206653 A1, whenadministered systemically in combination with metal-bindingglycoproteins, can reach immune privileged sites by penetrating theblood-brain barrier, the retina-blood barrier and theblood-cerebrospinal fluid barrier.

The invention is also based on the additional unexpected discovery thatthe administration of such PDC-glycoprotein combinations leads topreferential and persistent accumulation of the PDCs in malignant cellsand tissues.

The brain and eye are immune privileged sites protected by the bloodbrain barrier and blood retina barrier. Most biomolecules cross theblood-brain barrier by transmembrane diffusion whereby a low molecularweight and high lipid solubility are favored. Other drugs orphotosensitizer can cross the blood-brain barrier by the use ofsaturable transport systems.

Ruthenium and osmium are in the same group of the periodic table ofelements as Iron, Group 8, and share many of its characteristics.Rhodium is in neighboring Group 9. As Ruthenium and Osmium are in thesame group as Iron, they share many characteristics. For example,electronically, Ru (II) and OS (II) molecules readily bond with nitrogenand sulphur donor (this mechanisms is also relevant for induction of NO(Chatterjee D, Shome S, Jaiswal N, Banerjee P. Nitrite reductionmediated by the complex Ru III (EDTA). Dalton Trans. 2014 Sep. 28;43(36):13596-600) and may play a role in switching the Type II to Type Iphotochemistry) molecules that are abundantly found in many proteinswithin the body. For this reason transition metal complexes are able totake advantage of the body's ability to efficiently transport and uptakeof iron (Antonarakis E S, Emadi A. Ruthenium-based chemotherapeutics:are they ready for prime time? Cancer Chemother Pharmacol. 2010 May;66(1):1-9). The PSs are transported inside cancer cell (mitochondria) bybinding to endogenous transferrin (a glycoprotein mainly produced in theliver) via transferrin receptor (TfR); Bergamo A, Sava G. Rutheniumanticancer compounds: myths and realities of the emerging metal-baseddrugs. Dalton Trans. 2011 Aug. 21; 40(31):7817-23.

Rapidly dividing tumor cells have an increased demand for iron and thelevels of TfRs found on these cancerous cells are greatly increased. Thereceptor increase on cancerous cells has been document as two to twelvetimes that of healthy cells (Antonarakis E S, Emadi A. Ruthenium-basedchemotherapeutics: are they ready for prime time? Cancer ChemotherPharmacol. 2010 May; 66(1):1-9). This greatly increases the selectivityof the PSs as the majority of the dose is sequestered in canceroustissues, bypassing most healthy cells. This effect contributes to thelower toxicity that is associated to the ruthenium drugs in comparisonto platinum (Bruijnincx, Pieter C. A.; Sadler, Peter J. (2009).“Controlling platinum, ruthenium, and osmium reactivity for anticancerdrug design”. Advances in Inorganic Chemistry 61. p. 1).

Because iron is required for growth of cancer cells, we see anadditional benefit of competitive binding of PDCs to the transferrinreceptor on a tumor cell. In addition to the active uptake of PDCs, theuse of this mechanism in inhibiting both cell proliferation and HIF-1α.Angiogenesis under normoxic and hypoxic conditions may be of additionaltherapeutic use. Moreover, as cancer cells are generally growing andmultiplying much more rapidly than normal healthy cells, this creates anenvironment that is less oxygen rich due to the raised metabolic rate.When this is paired with the tendency of cancerous cells to containhigher levels of glutathione and a lower pH, a chemically reducingenvironment is created. Indeed, our data have shown an additive and evenpotentially synergistic role of glutathione in the mechanisms of cancercells kill by PDCs. The glutathione-mediated reduction is thought tooccur by mitochondrial proteins or microsomal single electron transferproteins, though it may also occur by trans-membrane electron transportsystems which reside outside the cell, implying that, due to localadministrations of PSs, the PS is still effective even if some quantityof the PS may leak/escape into intracellular space. In theory, it isalso possible for the Ruthenium compounds to be oxidized back to theirinactive form if they leave the cancerous environment and, hence, inaddition to a very strong photostability of PSs, this phenomenon mayalso contribute to additional treatment efficacy and safety.

During light activation in preferred embodiments of the invention, inaddition to PDT-induced inflammation, there is modification of tumorcell death and antigen presenting cells (“APC”) activation via thedanger-associated molecular patterns (“DAMPs”). The recognition ofmolecules released or expressed by dead, dying, injured, or stressed“antigenic”-apoptotic cells can elicit potent and tumor-specific immuneresponses. PDT-induced DAMPs emitted by dying cancer cells can elicitcancer antigen-directed anti-tumor short-term effects (6 to 8 weeks) anda long-term anti-tumor effect (>10 months) immunity. DAMP's stimulateimmune responses through dialogue with T lymphocytes (“Th”) cells,Natural Killer (“NK”) cells and APSs. Certain APSs, such as dendriticcells and macrophages are stimulated and actively trafficked duringPDT-induced “immunologic” cell death (“ICD”) by danger signallingpathways, which are instigated and regulated by a complex interplaybetween cellular stress signaling, reactive oxygen species (“ROS”)production and certain metabolic/biosynthetic processes (i.e.,autophagy, caspase activity and secretory pathway: calreticulin,Adenosine Tri-Phosphate (“ATP”), Heat Shock Proteins, High MobilityGroup Box 1, cytokines, etc).

Methods of the Invention

Methods of the invention comprise the use of metal-binding glycoproteinsas delivery vehicles for metal-based chemotherapeutic agents(preferably, PDCs) so as to facilitate delivery of the PDCs into abiological target. The methods are intended to provide enhanced safetyand/or efficacy relative to the delivery of metal-based PDCs without theaddition of exogenous metal-binding glycoproteins. PDCs delivered viathe inventive methods and compositions enjoy enhanced biophysical,biochemical and biomedical properties, such as the efficacy,tolerability, therapeutic efficacy and diagnostic properties of the PDCsin multi-wavelength photodynamic therapy in normoxic and hypoxicconditions.

Hence, methods of the invention comprise combining metal-containing PDCswith metal-binding glycoproteins and administering the combination to apatient in need of PDT at an immune privileged site.

The PDC and glycoprotein are preferably administered as a complex withina pharmaceutically acceptable dosage form. The dosage form can furthercomprise diluents, extenders, carriers and the like. The dosage form ispreferably a liquid, solid, gel or combination thereof. Suitable dosageforms include but are not limited to pills, tablets, capsules, eye dropsand injectable liquids. The dosage form can be administered orally,rectally, topically, parenterally or intravenously. Administration canbe systemic or localized (e.g., by injection into a tumor).

The PDC is preferably photoactive so as to generate reactive oxygenspecies. Photoactivation can be achieved by the application of lightfrom a light source. Suitable light sources include but are not limitedto lasers, light emitting diodes, fiber optics and lamps. The lightsource can be external to the patient's body with the light be appliedtransdermally. Alternatively, the light source can be placed within thepatient's body briefly (e.g, through an endoscope or fiber opticcatheter) or over an extended duration (e.g., as an implant).

In certain embodiments, the PDC can be activated by ionizing radiationin accordance with the teachings of U.S. Application 62/325,226, filedApr. 20, 2016. The ionizing radiation is preferably at least one ofX-rays and Gamma rays, and is preferably applied to treat targets atimmune privileged sites.

PDT dose parameters can be determined by a person of ordinary skill inthe art with an understanding of the dosimetric and biological factorsthat govern therapeutic variability. See, e.g., Rizvi et al. “PDT DoseParameters Impact Tumoricidal Durability and Cell Death Pathways in a 3DOvarian Cancer Model.” Photochemistry and photobiology. 2013;89(4):942-952.

Factors to be considered include but are not limited to the amount ofthe PDC at the target site, tissue oxygenation, the molar extinctioncoefficient of the PDC at a chosen wavelength of light to produce amaximum level of reactive oxygen species, target (e.g. tumor)localization, size, shape, vascular structure, etc. The following tablelists PDT parameters to be adjusted and provides preferred,non-exhaustive, values for said parameters.

PDT Parameter Value Wavelength (nm) 200-1000 or 400-950 or 500-950Fluence (J/cm²) 0.01 to 100,000 or 1 to 10,000 or 10 to 1,000 Irradiance(mW/cm²) 10 to 10,000 or 50 to 5,000 or 100 to 1,000 Irradiation Time(secs) 1 to 10,000 or 10 to 5,000 or 100 to 1,000 PDC Concentration 0.01to 6000 or 0.1 to 5000 or 1 to 2500 or (mg/kg body wt.) 10 to 1000 or 50to 500

In certain embodiments, the glycoprotein complex including themetal-containing PDC can be used as a carrier to transport other activepharmaceutical ingredients (APIs) to target tissues, such as immuneprivileged sites. Suitable APIs are not particularly limited, but mostpreferred are anti-neoplastic agents. The API need not contain atransition metal and need not be a PDC. Preferably, the PDC is achemotherapeutic compound and the API is an anti-neoplastic agentdifferent from the PDC.

For example, temozolomide attached via a linker to the glycoprotein-PDCcomplex could be administered to a cancer patient. The half-life oftemozolomide is approximately 1.8 hours. The half-life of the PDC candiffer from the API so that treatment is effective over an extendedperiod of time. The glycoprotein-PDC complex with the API will bepreferentially taken-up by the target tumor and cause less toxicity tothe benign tissue. The effects of the API can optionally be followed byPDT.

The present invention further relates to a method for preparing thecompounds of the present invention.

Compounds of the present teachings can be prepared in accordance withthe procedures outlined herein, from commercially available startingmaterials, compounds known in the literature, or readily preparedintermediates, by employing standard synthetic methods and proceduresknown to those skilled in the art. Standard synthetic methods andprocedures for the preparation of organic molecules and coordinationcomplexes and functional group transformations and manipulations can bereadily obtained from the relevant scientific literature or fromstandard textbooks in the field. It will be appreciated that wheretypical or preferred process conditions (i.e., reaction temperatures,times, mole ratios of reactants, solvents, pressures, etc.) are given,other process conditions can also be used unless otherwise stated.Optimum reaction conditions can vary with the particular reactants orsolvent used, but such conditions can be determined by one skilled inthe art by routine optimization procedures. Those skilled in the art oforganic and inorganic synthesis will recognize that the nature and orderof the synthetic steps presented can be varied for the purpose ofoptimizing the formation of the compounds described herein.

The preparation methods described herein can be monitored according toany suitable method known in the art. For example, product formation canbe monitored by spectroscopic means, such as nuclear magnetic resonancespectroscopy (e.g., 1H or 13C), infrared spectroscopy, spectrophotometry(e.g., UV-visible), mass spectrometry, or by chromatography such as highpressure liquid chromatography (HPLC), gas chromatography (GC),gel-permeation chromatography (GPC), or thin layer chromatography (TLC).

Metal-binding glycoproteins suitable for use in the invention arecapable of binding transition metals and delivering to a biologicaltarget said metals and other materials complexed with said metals. Themetal-binding glycoproteins are preferably capable of binding Group 8metals and/or Group 9 metals, and most preferably Ru, Os, Rh and Ir.Most preferred are the iron-binding glycoproteins transferrin,lactoferrin, ovotransferrin and melanotransferrin and variants thereof,with transferrin being most preferred. The glycoprotein can be purifiedfrom natural sources or can be from artificial sources. Thus, forexample, the glycoprotein in certain embodiments is a recombinanttransferrin, such as Apo-Transferrin or OPTIFERRIN, a recombinant humantransferrin available from InVitria, a division of Ventria Bioscience.See US 20120088729 Aa, Zhang et al., “Expression, purification, andcharacterization of recombinant human transferrin from rice (Oryzasativa L.).” Protein Expr Purif. 2010 November; 74(1):69-79. Epub 2010May 4, and Steere et al., “Biochemical and structural characterizationof recombinant human serum transferrin from rice (Oryza sativa L.).” JInorg Biochem. 2012 Jul. 11; 116C:37-44. OPTIFERRIN is a particularlypreferred glycorprotein as it increases the targeting and reduces thephotobleaching of the metal-glycoprotein complexes of the invention.

Biological targets of the invention are organisms, organs, tissuesand/or cells amenable to treatment with, and/or detection by, themetal-glycoprotein complexes of the invention. The targets arepreferably hyperproliferating cells, such as cancer and non-malignantlesions. Most preferably, the targets are immune privileged. Theinvention enables the treatment of targets across the blood-brain,blood-retina and blood-cerebrospinal fluid barriers.

Tf solution pre-mixed with PS solution (Ruthenium, Ruthenium-Rhodium andOsmium-based PSs) demonstrates evidence of metal-specific binding of thePS to Tf. The resulting complex has increased absorbance/molarextinction coefficient at long wavelengths (>600 nm), increased ROSproduction (generation of hydroxyl radical is potentiated to a muchgreater extent than that of singlet oxygen suggesting a switch to Type Iphotoreaction in the presence of transferrin), increased andpreferential uptake by cancer cells, increased efficacy of in vitro PDTaccompanied by a decrease in dark toxicity and consequently by increasedtherapeutic ratio, increased and selective uptake by cancer cells andtumor tissues and increased efficacy of in vivo PDT in visible and NIRlight. In certain embodiments, increased absorbance/molar extinctioncoefficient at wavelengths at or below 600 nm are achieved, whichembodiments are particularly suitable for excitation with green, blue,UV and X-ray radiation.

Ruthenium, Ruthenium-Rhodium and Osmium-based photosensitizers bind totransferrin demonstrating characteristic binding signatures.

Binding to transferrin changes chemical, physical and biomedicalcharacteristics of metal-based molecules and/or formulations, andinduces absorption by Ruthenium, Ruthenium-Rhodium and Osmium-basedphotosensitizers in red and NIR wavelengths where their absorptionwithout transferrin is negligible.

Irradiation-induced fluorescence of Ruthenium, Ruthenium-Rhodium andOsmium-based photosensitizers is increased in the presence oftransferrin, which may play a role in using metal-transferrin complexesin diagnostics.

Binding of Ruthenium, Ruthenium-Rhodium and Osmium-basedphotosensitizers to transferrin will increase their resistance tophotobleaching.

Binding of Ruthenium, Ruthenium-Rhodium and Osmium-basedphotosensitizers to transferrin will increase production of reactiveoxygen species in a cell free environment. This effect will increasewith the increase of transferrin concentration. Increased production ofhydroxyl radical suggests that the photosensitizers' photoeffect isswitched from Type II to Type I, which is essential for PDT treatment ofbulky hypoxic tumors.

Binding of Ruthenium, Ruthenium-Rhodium and Osmium-basedphotosensitizers to transferrin will increase their preferential uptakeby cancer cells.

In the presence of additional transferrin, Ruthenium, Ruthenium-Rhodiumand Osmium-based photosensitizers will demonstrate lesser dark toxicityin vitro (using cancer cell lines); PDT efficacy will increase. Thiswill result in the increase in therapeutic ratios of thephotosensitizers.

Binding of Ruthenium, Ruthenium-Rhodium and Osmium-basedphotosensitizers to additional transferrin will demonstrate PDT efficacyin hypoxic conditions absent without transferrin.

Blocking transferrin receptors with specific antibodies will eliminatefacilitating effect of transferrin on PDT effect.

Incubation of Ruthenium, Ruthenium-Rhodium and Osmium-basedphotosensitizers with transferrin prior to i.p. injection in vivo willincreases MTD50 indicating a decrease in the PSs toxicity.

Incubation of Ruthenium, Ruthenium-Rhodium and Osmium-basedphotosensitizers with transferrin prior to IP injection will increase invivo PDT efficacy in NIR (808 nm) and red (625 nm).

The invention will enable:

-   -   Selective delivery of Ruthenium, Ruthenium-Rhodium and        Osmium-based photosensitizers to immune privileged targets;    -   Increase in efficacy of the PS, especially in wavelength ranges        where the photosensitizers are otherwise ineffective (red, NIR);    -   PDT efficacy in hypoxia when the PSs are not effective without        Tf;    -   Improved safety, tolerability and efficacy of all metal-based        medicinal formulations that could be linked to Tf and/or        Tf-based substances, including beads and or liposomes; and    -   Improved diagnostic properties of metal based molecules.

The invention further encompasses the use of metal-glycoproteincomplexes of the invention to enhance uptake by cells of metal-basedpharmaceutical agents that are not light activated (e.g., RAPTA, NAMI,KP1019, RM-175).

Compositions of the Invention

The compositions of the invention comprise a metal-binding glycoproteinand a chemotherapeutic compound (e.g., a photodynamic compound)containing at least one transition metal, which is preferably a Group 8or 9 metal and is most preferably at least one of Ru, Rh and Os.

The chemotherapeutic compound is preferably at least one such compounddisclosed in WO 2013158550 A1, WO 2014145428 A2, U.S. Pat. No.6,962,910, U.S. Pat. No. 7,612,057, U.S. Pat. No. 8,445,475, U.S. Pat.No. 8,148,360 or US 20160206653 A1. Other chemotherapeutic compoundssuitable for use in the invention include but are not limited to RAPTA,NAMI, KP1019 and analogs thereof.

In certain embodiments, compositions of the invention comprisetransitional metal-based combinations that are optionally surrounded(directly and/or indirectly binded) by various ligands, such as incoordination compounds. In some of these embodiments, bridging ligandsare included, which are highly electron donating to the metal(s).

In all of the embodiments provided herein, examples of suitable optionalsubstituents are not intended to limit the scope of the claimedinvention. The compounds of the invention may contain any of thesubstituents, or combinations of substituents, provided herein.

The compositions of the invention are useful for the treatment anddiagnosis of disease states, particularly for the destruction ofinfectious organisms, hyperproliferating cells, and tumor cells. Thecompositions preferably included PDCs which (i) are metal-basedcoordination complexes, (ii) absorb ultraviolet (UV), visible, andinfrared (IR) (particularly, near infrared (NIR)) light and areactivated by wavelengths from UV to IR, particularly >800 nm, (iii) killhuman cancer cells in culture and in animals, and (iv) destroy bacteriaand antibiotic-resistant bacteria.

Compositions of the invention are also capable of destroyingmicroorganisms, such as Staphylococcus aureus (SA) andmethicillin-resistant S. aureus (MRSA), with activation by UV to IRlight, particularly red and NIR light.

The invention will be illustrated in more detail with reference to thefollowing Examples, but it should be understood that the presentinvention is not deemed to be limited thereto.

The Examples provided below provide representative methods for preparingexemplary compounds of the present invention. The skilled practitionerwill know how to substitute the appropriate reagents, starting materialsand purification methods known to those skilled in the art, in order toprepare the compounds of the present invention.

EXAMPLES Example 1

Transferrin (0.2 mg/mL) was incubated with different photosensitizers(10 uM) and the absorbance spectra were obtained. FIG. 1A shows anincrease in absorption by Ruthenium (TLD1433). FIG. 1B showsRuthenium-Rhodium (TLD143310) based PSs and FIG. 1C shows Osmium(TLDOsH2dppn) based PSs in the presence of transferrin. For eachphotosensitizer, the left plot shows the absolute difference inabsorbance with transferrin vs. without transferrin. The right plotshows relative increase in absorbance with transferrin vs. withouttransferrin. Note that a large relative increase in absorbance may beaccompanied by a small absolute difference and vice versa.

The binding of the photosensitizers to transferrin was accompanied by anincreased absorption in visible and NIR range, especially at longerwavelengths. It is noteworthy that negligible absorption of thephotosensitizers in red & NIR range became substantial upon binding ofthese photosensitizers to transferrin.

Example 2 Binding Signatures

Transferrin (0.2 mg/mL) was incubated with the photosensitizers (10 uM)and absorption spectra were obtained. FIGS. 2A, 2B and 2C showtransferrin binding to Ruthenium (TLD1433, FIG. 2A), Ruthenium-Rhodium(TLD143310, FIG. 2B) and Osmium (TLDOsH2dppn, FIG. 2C) basedphotosensitizers.

Binding of Ruthenium and Ruthenium-Rhodium-based photosensitizers totransferrin is characterized by a characteristic signature: an increasedpeak of absorption at 280 nm and a new peak in 400-500 nm range.Osmium-based photosensitizers demonstrate different binding signature:new peaks in 300-400 nm and 500-600 nm ranges.

Example 3 Fluorescence

Fluorescence of TLD1433 (10 uM) was measured quartz cuvettes without Tf(in water) and in the presence of 0.2 mg/mL Tf (in phosphate buffer+100mM NaCl, pH=7.0). Fluorescence was measured at different excitationwavelengths (380, 400, 450, 470, 500 nm).

TLD1433 demonstrates measurable fluorescence that is intensified in thepresence of transferrin. With no transferrin (FIG. 3A), maximalfluorescence was evoked at excitation wavelengths 380, 400 and 470 nm(emission maximum 624 nm). In the presence of transferrin (FIG. 3B),fluorescence was strongly increased, especially at 470 nm excitation,with emission maximum slightly shifted towards shorter wavelengths (619nm).

Detectable fluorescence of TLD1433 makes it useful for diagnosticpurposes (detection of selective uptake of TLD1433, preferably bycancerous tissues).

Example 4 Prophetic

FIGS. 4A and 4B

Fluorescence of TLD1433 (10 uM) was measured quartz cuvettes without Tf(in water) and in the presence of 0.2 mg/mL Tf (in phosphate buffer+100mM NaCl, pH=7.0). Fluorescence was measured at different excitationwavelengths (380, 400, 450, 470, 500 nm).

TLD143310 demonstrates measurable fluorescence that is intensified inthe presence of transferrin. With no transferrin (FIG. 4A), maximalfluorescence was evoked at excitation wavelengths 380, 400, 470 nm(emission maximum 650 nm). In the presence of transferrin (FIG. 4B),fluorescence was strongly increased, especially at 470 nm excitation,with emission maximum slightly shifted towards shorter wavelengths (637nm). Detectable fluorescence of TLD143310 makes it useful for diagnosticpurposes (detection of selective uptake of TLD143310, preferably bycancerous tissues).

Example 5 Prophetic

Fluorescence of TLDOsH2dppn (10 uM) was measured quartz cuvettes withoutTf (in water) and in the presence of 0.2 mg/mL Tf (in phosphatebuffer+100 mM NaCl, pH=7.0). Fluorescence was measured at differentexcitation wavelengths (380, 400, 450, 470, 500 nm).

TLDOsH2dppn demonstrates measurable fluorescence that is intensified inthe presence of transferrin. With no transferrin (FIG. 5A), maximalfluorescence was evoked at excitation wavelengths 380, 400, 470 nm(emission maximum 660 nm). In the presence of transferrin (FIG. 5B),fluorescence was strongly increased, especially at 470 nm excitation,with emission maximum slightly shifted towards shorter wavelengths (650nm).

Detectable fluorescence of TLDOsH2dppn makes it useful for diagnosticpurposes (detection of selective uptake of TLDOsH2dppn, preferably bycancerous tissues).

Example 6 Photobleaching

Photobleaching was measured: (a) at wavelength in visible range maximalfor each photosensitizer. This allows to estimate dynamics of amount ofunbleached photosensitizer during irradiation; and (b) at 530, 635 and808 nm. This allows estimating dynamics of unbleached photosensitizeravailable to exert PDT effect at these wavelengths as practically usefulfor PDT treatments.

Photosensitizers (10 uM) were dissolved in phosphate buffer+100 mM NaCl(pH=7.0) alone or in a buffer with 0.8 mg/mL of transferrin at a totalvolume of 1 mL. The mixture was then exposed to a 525 nm laser source(130 mW) and the absorbance at the maximal absorbance in visible range(432 nm for TLD1433, 425 nm for TLD143310 and 562 nm for TLDOsH2dppn)was measured at specific time points. The plot represents the absorbanceratio (absorbance of exposed samples/absorbance of unexposed sample) asa function of the number of incident photons per cm⁻². Decreasingabsorbance ratio signifies bleaching of the photosensitizer.

Effect of Transferrin on bleaching of Ruthenium (TLD1433, FIG. 6A),Ruthenium-Rhodium (TLD143310, FIG. 6B—Prophetic) and Osmium(TLDOsH2dppn, FIG. 6C—Prophetic)-based compounds at 525 nm.

In the presence of transferrin, bleaching of the photosensitizers wasreduced.

Example 7 Prophetic

Photosensitizers (10 uM) were dissolved in phosphate buffer+100 mM NaCl(pH=7.0) alone or in a buffer with 0.8 mg/mL of transferrin at a totalvolume of 1 mL. The mixture was then exposed to a 525 nm laser source(130 mW) and the absorbance at 530 nm was measured at specific timepoints. The plot represents the absorbance ratio (absorbance of exposedsamples/absorbance of unexposed sample) as a function of the number ofincident photons per cm⁻². Decreasing absorbance ratio signifiesbleaching of the photosensitizer.

Effect of Transferrin on bleaching of Ruthenium (TLD1433, FIG. 7A),Ruthenium-Rhodium (TLD143310, FIG. 7B) and Osmium (TLDOsH2dppn, FIG.7C)-based compounds: absorbance at 525 nm. In the presence oftransferrin, bleaching of the photosensitizers was reduced. Transferrinpartially protects photosensitizers from photobleaching.

Example 8 Prophetic

Photosensitizers (10 uM) were dissolved in phosphate buffer+100 mM NaCl(pH=7.0) alone or in a buffer with 0.8 mg/mL of transferrin at a totalvolume of 1 mL. The mixture was then exposed to a 525 nm laser source(130 mW) and the absorbance at 635 nm was measured at specific timepoints. The plot represents the absorbance ratio (absorbance of exposedsamples/absorbance of unexposed sample) as a function of the number ofincident photons per cm⁻². Decreasing absorbance ratio signifiesbleaching of the photosensitizer.

Effect of Transferrin on bleaching of Ruthenium (TLD1433, FIG. 8A),Ruthenium-Rhodium (TLD143310, FIG. 8B) and Osmium (TLDOsH2dppn, FIG. 8C)based compounds: absorbance at 635 nm. In the presence of transferrin,bleaching of the photosensitizers was reduced. Transferrin partiallyprotects photosensitizers form photobleaching.

Example 9 Prophetic

Photosensitizers (10 uM) were dissolved in phosphate buffer+100 mM NaCl(pH=7.0) alone or in a buffer with 0.8 mg/mL of transferrin at a totalvolume of 1 mL. The mixture was then exposed to a 525 nm laser source(130 mW) and the absorbance at 808 nm was measured at specific timepoints. The plot represents the absorbance ratio (absorbance of exposedsamples/absorbance of unexposed sample) as a function of the number ofincident photons per cm⁻². Decreasing absorbance ratio signifiesbleaching of the photosensitizer.

Effect of Transferrin on bleaching of Ruthenium (TLD1433, Panel A),Ruthenium-Rhodium (TLD143310, Panel B) and Osmium (TLDOsH2dppn, Panel C)based compounds: absorbance at 808 nm. In the presence of transferrin,bleaching of the photosensitizers was reduced. Transferrin partiallyprotects photosensitizers form photobleaching.

Example 10 ROS Production (Prophetic)

Table 1 shows singlet oxygen production by the Ruthenium (TLD1433),Ruthenium-Rhodium (TLD143310) and Osmium (TLDOsH2dppn)-based PS at 530nm & 1092 J cm−2 incident energy.

TABLE 1 TLD1433 TLD143310 TLDOsH2dppn Phosphate buffer no transferrin22471 21068 25937 0.8 mg/mL 45568 32816 42816 transferrin 1.6 mg/mL61568 55934 56372 transferrin 3.2 mg/mL 82652 751407 77219 transferrinIncomplete DMEM no transferrin 32398 21936 36391 0.8 mg/mL 53549 4213835127 transferrin 1.6 mg/mL 73657 52384 43927 transferrin 3.2 mg/mL95368 71573 61734 transferrin

Example 11 Prophetic

Table 2 shows hydroxyl radical production by the Ruthenium (TLD1433),Ruthenium-Rhodium (TLD143310) and Osmium (TLDOsH2dppn)-based PS at 530nm & 1092 J cm−2 incident energy.

TABLE 2 TLD1433 TLD143310 TLDOsH2dppn Phosphate buffer no transferrin31358 28030 25290 0.8 mg/mL transferrin 112047 70107 41037 1.6 mg/mLtransferrin 130308 121038 83034 3.2 mg/mL transferrin 165023 153096120307 Incomplete DMEM no transferrin 41034 30348 32934 0.8 mg/mLtransferrin 150237 60108 70308 1.6 mg/mL transferrin 182037 130025132309 3.2 mg/mL transferrin 190608 162027 164301

Example 12 Prophetic

Table 3 shows singlet oxygen production by the Ruthenium (TLD1433),Ruthenium-Rhodium (TLD143310) and Osmium (TLDOsH2dppn)-based PS at 635nm & 649 J cm−2 incident energy.

TABLE 3 TLD1433 TLD143310 TLDOsH2dppn Phosphate buffer no transferrin91374 10289 10392 0.8 mg/mL 53124 41301 42837 transferrin 1.6 mg/mL57621 68027 51832 transferrin 3.2 mg/mL 72218 75280 72938 transferrinIncomplete DMEM no transferrin 19929 9507 15109 0.8 mg/mL 39824 3193753931 transferrin 1.6 mg/mL 82438 70218 75293 transferrin 3.2 mg/mL95341 75317 81807 transferrin

Example 13 Prophetic

Table 4 shows hydroxyl radical production by the Ruthenium (TLD1433),Ruthenium-Rhodium (TLD143310) and Osmium (TLDOsH2dppn)-based PS at 635nm & 649 J cm−2 incident energy.

TABLE 4 TLD1433 TLD143310 TLDOsH2dppn Phosphate buffer no transferrin12608 15000 8804 0.8 mg/mL transferrin 71907 50328 25280 1.6 mg/mLtransferrin 82093 81867 45372 3.2 mg/mL transferrin 101293 85361 72316 9Incomplete DMEM no transferrin 18107 15280 12093 0.8 mg/mL transferrin98928 43623 32531 1.6 mg/mL transferrin 119307 72901 42806 3.2 mg/mLtransferrin 152704 93150 73624

Example 14 Prophetic

Table 5 shows singlet oxygen production by the Ruthenium (TLD1433),Ruthenium-Rhodium (TLD143310) and Osmium (TLDOsH2dppn)-based PS at 808nm & 4000 J cm−2 incident energy.

TABLE 5 TLD1433 TLD143310 TLDOsH2dppn Phosphate buffer no transferrin10621 9000 8000 0.8 mg/mL 41934 20000 10000 transferrin 1.6 mg/mL 5630530000 30000 transferrin 3.2 mg/mL 62861 50000 40000 transferrinIncomplete DMEM no transferrin 0 0 11907 0.8 mg/mL 1204 1245 25384transferrin 1.6 mg/mL 22394 12307 55631 transferrin 3.2 mg/mL 3602725193 62804 transferrin

Example 15

Table 6 shows hydroxyl radical production by the Ruthenium (TLD1433),Ruthenium-Rhodium (TLD143310) and Osmium (TLDOsH2dppn)-based PS at 808nm & 4000 J cm−2 incident energy.

TABLE 6 TLD1433 TLD143310 TLDOsH2dppn Phosphate buffer no transferrin15610 12305 8931 0.8 mg/mL transferrin 22967 15034 13408 1.6 mg/mLtransferrin 56390 31384 20392 3.2 mg/mL transferrin 61832 63904 56094Incomplete DMEM no transferrin 503 427 14237 0.8 mg/mL transferrin 701714 42305 1.6 mg/mL transferrin 20357 12397 77024 3.2 mg/mL transferrin41395 23297 102864

Example 16 Prophetic

The photosensitizers were diluted in phosphate buffer (pH=7.0)+100 mMNaCl or incomplete DMEM. The irradiation was performed in 96-well plates(100 uL working volume). Generation of singlet oxygen was measured byfluorescence signal of SOG indicator, generation of hydroxyl radical byfluorescence signal of HPF indicator. The signal presented is a resultof subtraction of the signal in the presence of scavengers (NaN₃ forsinglet oxygen and DMTU for hydroxyl radical) from the totalfluorescence signal.

Ruthenium (TLD1433), Ruthenium-Rhodium (TLD143310) and Osmium(TLDOsH2dppn) based photosensitizers produce reactive oxygen species(ROS) under 525 nm (0.250 mW cm−2), 650 nm (0.119 mW cm−2) and NIR (808nm, 747 mW cm−2) irradiation.

In the presence of transferrin, generation of both singlet oxygen andhydroxyl radical is potentiated in a dose-dependent manner: higherconcentration of transferrin induces greater generation of ROS.Generation of hydroxyl radical is potentiated to a much greater extentthan that of singlet oxygen suggesting a switch to Type I photoreactionin the presence of transferrin.

Example 17 Intracellular Uptake (Prophetic)

Cell culture (U87 cells) was exposed to 200 uM of the photosensitizerwithout additional transferrin and in the presence of 0.8 mg/mLtransferrin for 4 hours. The incubation mix was washed away and thecells were collected, counted, and lysed. Lysed solution was dissolvedin nitric acid and appropriate metals concentration was measured usinginductively coupled plasma mass spectrometry. The metals concentrationis directly proportional to the concentration of the photosensitizer.

FIGS. 10A, 10B and 10C show transferrin facilitated uptake of Ruthenium(TLD1433, FIG. 10A), Ruthenium-Rhodium (TLD143310, FIG. 10B) and Osmium(TLDOsH2dppn, FIG. 10C) based photosensitizers into cancer cells.

Cellular accumulation of the photosensitizers was higher in the presenceof transferrin. Since tumor cells have been shown to have higherexpression of membrane transferrin receptor, these results show that thementioned photosensitizers can preferentially accumulate in tumor cells.

Example 18 Uptake by Normal Cells (Prophetic)

Cell culture (normal human fibroblasts) was exposed to 200 uM of thephotosensitizer without additional transferrin and in the presence of0.8 mg/mL transferrin for 4 hours. The incubation mix was washed awayand the cells were collected, counted, and lysed. Lysed solution wasdissolved in nitric acid and appropriate metals concentration wasmeasured using inductively coupled plasma mass spectrometry. The metalsconcentration is directly proportional to the concentration of thephotosensitizer.

FIGS. 11A, 11B and 11C show transferrin facilitated uptake of Ruthenium(TLD1433, FIG. 11A), Ruthenium-Rhodium (TLD143310, FIG. 11B) and Osmium(TLDOsH2dppn, FIG. 11C) based photosensitizers into normal cells.Cellular accumulation of the photosensitizers was higher in the presenceof transferrin. However, the effect of transferrin on the uptake is to amuch lesser extent than for cancer cells indicating uptake improvementpreferentially in cancer cells.

Example 19 In Vitro—Dark Toxicity

The photosensitizer was pre-mixed with 0.4 mg/mL human transferrin andincubated for 1 hour at 37° C. In control group (no additionaltransferrin), equivalent volume of no-transferrin medium was added. Thecells were subsequently incubated with the pre-mixes for 30 minutes(FIG. 12A) or 90 minutes (FIG. 12B). After that, the medium was replacedwith a fresh one (without photosensitizer and transferrin). On the nextday (21 hours post-irradiation), viability of the cells was measuredusing Presto Blue viability assay, and percent of cell kill wascalculated.

The presence of transferrin decreases dark toxicity (photosensitizeralone) of Ruthenium-based photosensitizers (TLD1433) on AY27 cancer cellline. Transferrin decreases dark toxicity of TLD1433. This contributesto an increased safety of PDT treatment in the presence of transferrin.Together with the evidence of binding of TLD1433 to transferrin, theseresults suggest facilitated uptake of TLD1433 into cells in the presenceof transferrin. Increase in PDT efficacy together with the decrease indark toxicity suggests an increase in therapeutic ratio of TLD1433 whenit is used mixed with transferrin.

Example 20 Prophetic

The photosensitizer was pre-mixed with 0.4 mg/mL human transferrin andincubated for 1 hour at 37° C. In control group (no additionaltransferrin), equivalent volume of no-transferrin medium was added. Thecells were subsequently incubated with the pre-mixes for 30 minutes.After that, the medium was replaced with a fresh one (withoutphotosensitizer and transferrin). On the next day (21 hourspost-irradiation), viability of the cells was measured using Presto Blueviability assay, and percent of cell kill was calculated.

The presence of transferrin decreases dark toxicity (photosensitizeralone) of Ruthenium-Rhodium (TLD143310, FIG. 13A) and Osmium(TLDOsH2dppn, FIG. 13B) based photosensitizers on AY27 cancer cell line.Transferrin decreases dark toxicity of the photosensitizers. Thiscontributes to an increased safety of PDT treatment in the presence oftransferrin.

Example 21 In Vitro PDT Effect

The photosensitizer was pre-mixed with 0.4 mg/mL human transferrin andincubated for 1 hour at 37° C. In control group (no additionaltransferrin), equivalent volume of no-transferrin medium was added. Thecells were subsequently incubated with the pre-mixes for 30 minutes(FIG. 14A) or 90 minutes (FIG. 14B). After that, the medium was replacedwith a fresh one (without photosensitizer and transferrin) and the cellswere irradiated. On the next day (21 hours post-irradiation), viabilityof the cells was measured using Presto Blue viability assay, and percentof cell kill was calculated. The PDT effect shown is a result ofsubtraction of “photosensitizer alone” and “light alone” cell kill fromthe total PDT cell kill.

The presence of transferrin increases PDT effect (635 nm, 90 J cm−2) ofRuthenium-based photosensitizers (TLD1433) on AY27 cancer cell line.Transferrin potentiates PDT effect of the photosensitizer even at shortloading time (30 minutes). This allows for safer PDT treatment due toshorter treatment time and the use of lower concentrations of thephotosensitizers. Together with the evidence of binding of TLD1433 totransferrin, these results suggest facilitated uptake of TLD1433 intocells in the presence of transferrin.

Example 22 Prophetic

The photosensitizer was pre-mixed with 0.4 mg/mL human transferrin andincubated for 1 hour at 37° C. In control group (no additionaltransferrin), equivalent volume of no-transferrin medium was added. Thecells were subsequently incubated with the pre-mixes for 30 minutes.After that, the medium was replaced with a fresh one (withoutphotosensitizer and transferrin) and the cells were irradiated. On thenext day (21 hours post-irradiation), viability of the cells wasmeasured using Presto Blue viability assay, and percent of cell kill wascalculated. The PDT effect shown is a result of subtraction of“photosensitizer alone” and “light alone” cell kill from the total PDTcell kill.

The presence of transferrin increases PDT effect (635 nm, 90 J cm−2) ofRuthenium-Rhodium (TLD143310, FIG. 15A) and Osmium (TLDOsH2dppn, FIG.15B) based photosensitizers on AY27 cancer cell line.

Transferrin potentiates PDT effect of the photosensitizers even at shortloading time (30 minutes). This allows for safer PDT treatment due toshorter treatment time and the use of lower concentrations of thephotosensitizers. Together with the evidence of binding of thephotosensitizers to transferrin, these results suggest facilitateduptake of the photosensitizers into cells in the presence oftransferrin.

Increase in PDT efficacy together with the decrease in dark toxicitysuggests an increase in therapeutic ratio of Ruthenium (TLD1433),Ruthenium-Rhodium (TLD143310) and Osmium (TLDOsH2dppn) basedphotosensitizers when they are pre-mixed with transferrin.

Example 23 Prophetic

The photosensitizer was pre-mixed with 0.4 mg/mL human transferrin andincubated for 1 hour at 37° C. In control group (no additionaltransferrin), equivalent volume of no-transferrin medium was added. Thecells were subsequently incubated with the pre-mixes for 30 minutes.After that, the medium was replaced with a fresh one (withoutphotosensitizer and transferrin) and the cells were irradiated. On thenext day (21 hours post-irradiation), viability of the cells wasmeasured using Presto Blue viability assay, and percent of cell kill wascalculated. The PDT effect shown is a result of subtraction of“photosensitizer alone” and “light alone” cell kill from the total PDTcell kill.

The presence of transferrin increases PDT effect (530 nm, 90 J cm−2) ofRuthenium-Rhodium (TLD143310, Panel A) and Osmium (TLDOsH2dppn, Panel B)based photosensitizers on AY27 cancer cell line. Transferrin potentiatesPDT effect of the photosensitizers even at short loading time (30minutes). This allows for safer PDT treatment due to shorter treatmenttime and the use of lower concentrations of the photosensitizers.Together with the evidence of binding of the photosensitizers totransferrin, these results suggest facilitated uptake of thephotosensitizers into cells in the presence of transferrin.

Example 24

Pre-mixing with transferrin increases therapeutic ratio of Ruthenium(TLD1433), Ruthenium-Rhodium (TLD143310) and Osmium (TLDOsH2dppn) basedphotosensitizers, due to the increase in PDT efficacy together with thedecrease in dark toxicity. See Table 7.

TABLE 7 Therapeutic Ratio HT1376 cells U87 cells AY27 cells No No Notransferrin Transferrin transferrin Transferrin transferrin Transferrin530 nm, 90 J cm−2 TLD1433 19024.1 50938.4 27557.1 65002.3 17356.257324.1 (Prophetic) (Prophetic) (Prophetic) (Prophetic) (Prophetic)TLD143310 421.9 835.7 495.1 1022.6 473.1 934.5 (Prophetic) (Prophetic)(Prophetic) (Prophetic) (Prophetic) TLDOsH2dppn 1548.2 4963.8 1307.35238.0 1639.8 5531.9 (Prophetic) (Prophetic) (Prophetic) (Prophetic)(Prophetic) 650 nm, 90 J cm−2 TLD1433 50.4 100.8 102.6 405.1 79.2 393.7(Prophetic) (Prophetic) (Prophetic) (Prophetic) (Prophetic) TLD14331028.1 52.7 16.3 50.2 25.1 59.4 (Prophetic) (Prophetic) (Prophetic)(Prophetic) (Prophetic) TLDOsH2dppn 37.7 61.9 15.3 50.3 28.7 61.7(Prophetic) (Prophetic) (Prophetic) (Prophetic)

Example 25 In Vitro Hypoxia (Prophetic)

The cells were incubated for 90 minutes in premix of the photosensitizerand 0.4 mg/mL human transferrin (or without transferrin, as a controlgroup) as described above for in vitro PDT. Dark toxicity and PDT effectof the photosensitizers on AY27 cell line under 635 nm (90 J cm−2)irradiation in the absence and presence of transferrin in normoxic(FIGS. 17A, 17B) and hypoxic (0.5-0.1% O₂, FIGS. 17C, 17D) conditions.The cells were incubated with the photosensitizer and transferrin for 90minutes. After that, the cells were irradiated and the medium wasreplaced with a fresh one (without photosensitizer and transferrin). Onthe next day (21 hours post-irradiation), viability of the cells wasmeasured using Presto Blue viability assay, and percent of cell kill wascalculated. The PDT effect shown is a result of subtraction of“photosensitizer alone” and “light alone” cell kill from the total PDTcell kill.

The presence of transferrin ensured PDT effect (635 nm, 90 J cm−2) ofRuthenium (TLD1433) based photosensitizers on AY27 cancer cell lineunder hypoxic conditions. Transferrin induces PDT activity in hypoxia,which is not observed in the absence of transferrin. The presence oftransferrin, therefore, allows for a greater efficacy of theRuthenium-based photosensitizers during PDT treatment of bulky hypoxictumors.

Example 26 Prophetic

The cells were incubated for 90 minutes in premix of the photosensitizerand 0.4 mg/mL human transferrin (or without transferrin, as a controlgroup) as described above for in vitro PDT. Dark toxicity and PDT effectof the photosensitizers on AY27 cell line under 635 nm (90 J cm−2)irradiation in the absence and presence of transferrin in normoxic(FIGS. 18A, 18B) and hypoxic (0.5-0.1% O₂, FIGS. 18C, 18D) conditions.The cells were incubated with the photosensitizer and transferrin for 90minutes. After that, the cells were irradiated and the medium wasreplaced with a fresh one (without photosensitizer and transferrin). Onthe next day (21 hours post-irradiation), viability of the cells wasmeasured using Presto Blue viability assay, and percent of cell kill wascalculated. The PDT effect shown is a result of subtraction of“photosensitizer alone” and “light alone” cell kill from the total PDTcell kill.

The presence of transferrin ensured PDT effect (635 nm, 90 J cm−2) ofRuthenium-Rhodium (TLD143310) based photosensitizers on AY27 cancer cellline under hypoxic conditions. Transferrin induces PDT activity inhypoxia, which is not observed in the absence of transferrin. Thepresence of transferrin, therefore, allows for a greater efficacy of theRuthenium-Rhodium-based photosensitizers during PDT treatment of bulkyhypoxic tumors.

Example 27 Prophetic

The cells were incubated for 90 minutes in premix of the photosensitizerand 0.4 mg/mL human transferrin (or without transferrin, as a controlgroup) as described above for in vitro PDT. Dark toxicity and PDT effectof the photosensitizers on AY27 cell line under 635 nm (90 J cm−2)irradiation in the absence and presence of transferrin in normoxic(FIGS. 19A, 19B) and hypoxic (0.5-0.1% O₂, FIGS. 19C, 19D) conditions.The cells were incubated with the photosensitizer and transferrin for 90minutes. After that, the cells were irradiated and the medium wasreplaced with a fresh one (without photosensitizer and transferrin). Onthe next day (21 hours post-irradiation), viability of the cells wasmeasured using Presto Blue viability assay, and percent of cell kill wascalculated. The PDT effect shown is a result of subtraction of“photosensitizer alone” and “light alone” cell kill from the total PDTcell kill.

The presence of transferrin ensured PDT effect (635 nm, 90 J cm−2) ofOsmium (TLDOsH2dppn) based photosensitizers on AY27 cancer cell lineunder hypoxic conditions.

Transferrin induces PDT activity in hypoxia, which is not observed inthe absence of transferrin. The presence of transferrin, therefore,allows for a greater efficacy of the Osmium-based photosensitizersduring PDT treatment of bulky hypoxic tumors.

Example 28 Transferrin Receptor Antibodies (Prophetic)

The cells were incubated with anti-transferrin receptor antibody. Thephotosensitizer was pre-mixed in parallel with 0.4 mg/mL humantransferrin and incubated for 1 hour at 37° C. In control group (noadditional transferrin), equivalent volume of no-transferrin medium wasadded. The cells were washed of the excess of antibody (the cellsincubated in medium without antibody served as comparison groups) andincubated with the pre-mixes for 30 minutes. After that, the medium wasreplaced with a fresh one (without photosensitizer and transferrin) andthe cells were irradiated. On the next day (21 hours post-irradiation),viability of the cells was measured using Presto Blue viability assay,and percent of cell kill was calculated. The PDT effect shown is aresult of subtraction of “photosensitizer alone” and “light alone” cellkill from the total PDT cell kill. Dark toxicity is shown on FIG. 20A,PDT effect is shown on FIG. 20B.

Blocking of transferrin receptors of AY27 cancer cells prevents decreasein dark toxicity and facilitation of PDT effect (635 nm, 90 J cm−2) ofRuthenium (TLD1433) based photosensitizers. The results support the roleof transferrin-mediated uptake of Ruthenium-based photosensitizers indecrease of their dark toxicity and facilitation of their PDT effect.

Example 29 Prophetic

The cells were incubated with anti-transferrin antibody. Thephotosensitizer was pre-mixed in parallel with 0.4 mg/mL humantransferrin and incubated for 1 hour at 37° C. In control group (noadditional transferrin), equivalent volume of no-transferrin medium wasadded. The cells were washed of the excess of antibody (the cellsincubated in medium without antibody served as comparison groups) andincubated with the pre-mixes for 30 minutes. After that, the medium wasreplaced with a fresh one (without photosensitizer and transferrin) andthe cells were irradiated. On the next day (21 hours post-irradiation),viability of the cells was measured using Presto Blue viability assay,and percent of cell kill was calculated. The PDT effect shown is aresult of subtraction of “photosensitizer alone” and “light alone” cellkill from the total PDT cell kill. Dark toxicity is shown on FIG. 21A,PDT effect is shown on FIG. 21B.

Blocking of transferrin receptors of AY27 cancer cells prevents decreasein dark toxicity and facilitation of PDT effect (635 nm, 90 J cm−2) ofRuthenium-Rhodium (TLD143310) based photosensitizers. The resultssupport the role of transferrin-mediated uptake of Ruthenium-Rhodiumbased photosensitizers in decrease of their dark toxicity andfacilitation of their PDT effect.

Example 30 Prophetic

The cells were incubated with anti-transferrin antibody. Thephotosensitizer was pre-mixed in parallel with 0.4 mg/mL humantransferrin and incubated for 1 hour at 37° C. In control group (noadditional transferrin), equivalent volume of no-transferrin medium wasadded. The cells were washed of the excess of antibody (the cellsincubated in medium without antibody served as comparison groups) andincubated with the pre-mixes for 30 minutes. After that, the medium wasreplaced with a fresh one (without photosensitizer and transferrin) andthe cells were irradiated. On the next day (21 hours post-irradiation),viability of the cells was measured using Presto Blue viability assay,and percent of cell kill was calculated. The PDT effect shown is aresult of subtraction of “photosensitizer alone” and “light alone” cellkill from the total PDT cell kill. Dark toxicity is shown in FIG. 22A,PDT effect is shown on FIG. 22B.

Blocking of transferrin receptors of AY27 cancer cells prevents decreasein dark toxicity and facilitation of PDT effect (635 nm, 90 J cm−2) ofOsmium (TLDOsH2dppn) based photosensitizers. The results support therole of transferrin-mediated uptake of Osmium-based photosensitizers indecrease of their dark toxicity and facilitation of their PDT effect.

Example 31 In Vivo Tissue Uptake (Prophetic)

Transferrin was dissolved in 40% PG in phosphate buffer (pH=7.0)+100 mMNaCl (0.1 mg/100 uL) and added to the photosensitizer solution (toachieve 10 mg/kg dose). The mixture was incubated for 1 hour prior toinjection, and 100 uL of the mixture was injected i.v. to each animal.

Mixing of the Ruthenium based photosensitizers (TLD1433) withtransferrin (1 mg/kg) increases uptake of the photosensitizer intotissues. The presence of transferrin considerably improves selectivityof the uptake into tumors as compared to normal muscle tissue (FIGS. 23Aand 23B).

Example 32 Prophetic

Transferrin was dissolved in 40% PG in phosphate buffer (pH=7.0)+100 mMNaCl (0.1 mg/100 uL) and added to the photosensitizer solution (toachieve 10 mg/kg dose). The mixture was incubated for 1 hour prior toinjection, and 100 uL of the mixture was injected i.v. to each animal.

Mixing of the Ruthenium-Rhodium based photosensitizers (TLD143310) withtransferrin (1 mg/kg) increases uptake of the photosensitizer intotissues. The presence of transferrin considerably improves selectivityof the uptake into tumors as compared to normal muscle tissue (FIGS. 24Aand 24B).

Example 33 Prophetic

Transferrin was dissolved in 40% PG in phosphate buffer (pH=7.0)+100 mMNaCl (0.1 mg/100 uL) and added to the photosensitizer solution (toachieve 10 mg/kg dose). The mixture was incubated for 1 hour prior toinjection, and 100 uL of the mixture was injected i.v. to each animal.

Mixing of the Osmium based photosensitizers (TLDOsH2dppn) withtransferrin (1 mg/kg) increases uptake of the photosensitizer intotissues. The presence of transferrin considerably improves selectivityof the uptake into tumors as compared to normal muscle tissue (FIGS. 25Aand 25B).

Example 34 In Vivo Toxicity

Mice were injected (i.p.) with escalating doses of the photosensitizerswith and without transferrin. Mixing of the photosensitizers withtransferrin increased MTD which suggests a decrease in toxicity of thephotosensitizers. For Ruthenium-based TLD1433, complete mouse survivalwas observed at 125 mg/kg; higher doses are currently being tested.

Maximum tolerated doses (MTD) of Ruthenium (TLD1433), Ruthenium-Rhodium(TLD143310) and Osmium (TLDOsH2dppn) based photosensitizers increaseswith transferrin are shown in Table 8 below.

TABLE 8 MTD Dose (mg/kg) With transferrin Photosensitizer No transferrin(1 mg/kg) TLD1433 100 >125 TLD143310 150 200 (Prophetic) (Prophetic)TLDOsH2dppn 50 100 (Prophetic)

Considering the results of in vitro and in vivo experiments combinedRuthenium, Ruthenium-Rhodium and Osmium-based photosensitizers must bemixed with transferrin for patients treatment not only to improve PDTeffect but also to decrease toxicity of the photosensitizers.

Example 35 In Vivo PDT Effect

Subcutaneous tumors were grown in mouse thighs and PDT (808 nm, 600Jcm⁻²) was performed when tumor reached 5×5 mm in size. Mice weresacrificed if tumor continued to grow after PDT treatment and reached10×10 mm. Controls had a maximum survival of 10 days.

Transferrin increases mouse survival after PDT treatment withRuthenium-based photosensitizer (TLD1433). The number of tumor freeanimals greatly increased when transferrin was added to Ruthenium-basedTLD1433 before injection (in our data currently 8 days after PDTtreatment, previous data has shown tumors will not reappear after thistime, 70 days is assumed to remain tumor free).

Example 36 Prophetic

Subcutaneous tumors were grown in mouse thighs and PDT (808 nm, 600Jcm⁻²) was performed when tumor reached 5×5 mm in size. Mice weresacrificed if tumor continued to grow after PDT treatment and reached10×10 mm. Controls had a maximum survival of 10 days.

Transferrin increases mouse survival after PDT treatment withRuthenium-Rhodium-based photosensitizer (TLD143310) and Osmium-based(TLDOsH2dppn) photosensitizers. The number of tumor free animals greatlyincreased when transferrin was added to Ruthenium-Rhodium-basedphotosensitizer (TLD143310) and Osmium-based (TLDOsH2dppn)photosensitizers before injection. See Table 9 below.

TABLE 9 Survival by day 80 Without transferrin With transferrin Mixedmetal TLD143310 35% 70% (Ruthenium- (Prophetic) (Prophetic)Rhodium)-based Osmium-based TLDOsH2dppn 40% 80% (Prophetic) (Prophetic)

Example 36

Subcutaneous tumors were grown in mouse thighs and PDT (635 nm, 192Jcm⁻²) was performed when tumor reached 5×5 mm in size. Mice weresacrificed if tumor continued to grow after PDT treatment and reached10×10 mm. Controls had a maximum survival of 10 days.

Transferrin increases mouse survival after PDT treatment. The number oftumor free animals greatly increased when transferrin was added toTLD1433 before injection (expected results). See FIG. 27.

Example 37

Subcutaneous tumors were grown in mouse thighs and PDT (635 nm, 192Jcm⁻²) was performed when tumor reached 5×5 mm in size. Mice weresacrificed if tumor continued to grow after PDT treatment and reached10×10 mm. Controls had a maximum survival of 10 days.

Transferrin increases mouse survival after PDT treatment withRuthenium-Rhodium-based photosensitizer (TLD143310) and Osmium-based(TLDOsH2dppn) photosensitizers. The number of tumor free animals greatlyincreased when transferrin was added to Ruthenium-Rhodium-basedphotosensitizer (TLD143310) and Osmium-based (TLDOsH2dppn)photosensitizers before injection. See Table 10.

TABLE 10 Survival by day 80 Without transferrin With transferrin Mixedmetal TLD143310 41% 65% (Ruthemium- (Prophetic) (Prophetic)Rhodium)-based Osmium-based TLDOsH2dppn 35% 75% (Prophetic) (Prophetic)

Example 38

Subcutaneous tumors were grown in mouse thighs and PDT (525 nm, 90Jcm⁻²) was performed when tumor reached 5×5 mm in size. Mice weresacrificed if tumor continued to grow after PDT treatment and reached10×10 mm. Controls had a maximum survival of 10 days.

Transferrin increases mouse survival after PDT treatment withRuthenium-Rhodium-based photosensitizer (TLD143310) and Osmium-based(TLDOsH2dppn) photosensitizers. The number of tumor free animals greatlyincreased when transferrin was added to Ruthenium-Rhodium-basedphotosensitizer (TLD143310) and Osmium-based (TLDOsH2dppn)photosensitizers before injection. See Table 11.

TABLE 11 Survival by day 80 Without transferrin With transferrin Mixedmetal TLD143310 48% 85% (Ruthenium- (Prophetic) (Prophetic)Rhodium)-based Osmium-based TLDOsH2dppn 37% 78% (Prophetic) (Prophetic)

Example 39

The optical density of TLD1433 was measured before and after theaddition of either OPTIFERRIN (available from InVitria, a division ofVentria Bioscience) or human apo-transferrin (Sigma). As shown in FIG.28, the TLD1433-OPTIFERRIN complex showed a substantial increase inoptical density. Furthermore, the complex showed novel absorption in thered (600 nm) and near infrared (800 nm) wavelengths. The optical densityof the OPTIFERRIN complex was comparable to that of the humanapo-transferrin complex. This finding highlights the potential of usingOPTIFERRIN to increase the efficacy of Theralase's TLD1433photosensitizer. The increase in optical density will translate to ahigher production of reactive oxygen species during PDT treatment. Theabsorption in red and near infrared light will allow for novel treatmentapplications for different tumors.

Example 40

The optical density of Optiferrin and human apo-transferrin was measuredbefore and after TLD1433 addition. Transferrin binding to iron ischaracterized by an increase in absorption at 275 nm and 450 nm. Muchlike human apo-transferrin, Optiferrin shows this characteristic opticaldensity increase after TLD1433 addition, showing direct binding ofTLD1433 to Optiferrin. See FIG. 29. Direct binding of Optiferrin toTLD1433 can be utilized in the treatment of transferrin receptor richtumors, where the Optiferrin+TLD1433 complex can be administered toachieve preferential uptake of TLD1433 in cancer cells.

Example 41

The bleaching of TLD1433 alone and incubated with Optiferrin or humanapo-transferrin in response to 525 nm (green) light was measured.Bleaching was measured by the decrease in the optical density at 425 nmin response to irradiation to 525 nm light, which was normalized bydividing it by the 425 nm optical density of the unexposed sample.Optical density ratio of 1 signifies no bleaching. Bleaching of TLD1433results in the ratio decreasing towards 0. Both Optiferrin and humanapo-transferrin reduced the bleaching of TLD1433, with Optiferrin havinga stronger effect. See FIG. 30.

Example 42 Optiferrin Binding with TLD1433 (14C TH2, GS6-22, andGS6-81G) and TH1 (Sky Blue)

Experiments demonstrated that the following photosensitizers mimic thebinding signatures of Fe+Tf binding, but with Optiferrin: TLD1433GS6-22, GS6-81G, TH2 QR-100, and TH1 lot no. 1411459022. See FIG. 31.

Example 43 Photobleaching Prevention Over 200 J with TLD1433

10 uM TLD1433 GS6-22 was photobleached with 200 J of green light (525nm) and its OD was measured at 0, 1, 2, 5, 10, 20, 50, 100, and 200Joules. See FIG. 32, which shows the ratio of Molar ExtinctionCoefficient increase or decrease at 350 nm, 400 nm, and 600 nm betweenthe initial time with 0 Joules, and the final time with 200 Joules.

Example 44 TLD1433 Infrared Absorbance Stability

Experiments demonstrated that the OD of TLD1433 is better maintained inthe infrared with the addition of 0.8 g/L (or 10 uM) Optiferrin overtime. See FIG. 33.

Example 45 Optiferrin Incubation and the Maintenance of PhotosensitiserTLD1433 Absorbance

The incubation duration was examined to see how well a dark sample of 10uM TLD1433 would absorb with the addition of Optiferrin. See FIG. 34. Asseen previously this undoubtedly helps prevent TLD1433 fromphotobleaching.

Example 46 In Vivo PDT in Rat Glioblastoma

A four-month old rat was injected in the cerebral cortex with 5000 RG2cells (rat glioblastoma tumor model). Eleven days later, the rat wasinjected with TLD1433 (5 mg/kg) with human apo-transferrin. Thestructure of TLD1433 is shown below:

PDT was administered 24 hours later. Light source and fluence: 808 nmwavelength, 300 J/cm² delivered at 100 mW. The number of photonsabsorbed was 2.8×10¹⁷ photons/cm³. The rat survived 13 days post PDTtreatment.

The effect of PDT on tumor size is shown in FIGS. 35A-H. FIG. 35A is aT1 weighted MRI and FIG. 35B is a T2 weighted MRI of the rat 6 daysafter tumor cell injection. FIG. 35C is a T1 weighted MRI and FIG. 35Dis a T2 weighted MRI of the rat 10 days after tumor cell injection. FIG.35E is a T1 weighted MRI and FIG. 35F is a T2 weighted MRI of the rat 3days after PDT. FIG. 35G is a T1 weighted MRI and FIG. 35H is a T2weighted MRI of the rat 8 days after PDT.

Example 47 In Vivo PDT in Rat Glioblastoma

A five-month old rat was injected in the cerebral cortex with 5000 RG2cells. Eleven days later, the rat was injected with TLD1433 (5 mg/kg)with human apo-transferrin. PDT was administered 24 hours later. Lightsource and fluence: 808 nm wavelength, 600 J/cm² delivered at 200 mW.The number of photons absorbed was 4.41×10¹⁸ photons/cm³. The rat hasnot died. At the time of the drafting of this text, the rat had survived25 days post PDT treatment. We did not notice any behavioral orcognitive changes in the PDT treated rats of Examples 46 or 47.

The example demonstrates a significant survival benefit compared to 8days post PDT survival with aminolevulinic acid (ALA) mediated PDT inthis aggressive tumor model. It is a very aggressive tumor model becausesurvival of untreated controls is only 4 days.

The effect of PDT on tumor size is shown in FIGS. 36A-J. FIG. 36A is aT1 weighted MRI and FIG. 36B is a T2 weighted MRI of the rat 8 daysafter tumor cell injection. The tumor shown in these figures measuredabout 0.84 mm in diameter.

FIG. 36C is a T1 weighted MRI and FIG. 36D is a T2 weighted MRI of therat 10 days after tumor cell injection. The tumor shown in these figuresmeasured about 1.44 mm in diameter.

FIG. 36E is a T1 weighted MRI and FIG. 36F is a T2 weighted MRI of therat 3 days after PDT. It is difficult to measure the size of tumor 3days post PDT, as these images show extensive inflammatory response totreatment depicted by edema.

FIG. 36G is a T1 weighted MRI and FIG. 36H is a T2 weighted MRI of therat 10 days after PDT. FIG. 36I is a T1 weighted MRI and FIG. 36J is aT2 weighted MRI of the rat 20 days after PDT. There no tumor detectableby MRI imaging. No tissue is taking up contrast agent, suggestingcomplete destruction of the tumor.

Example 48 ICP-MS Studies

Inductively coupled plasma mass spectrometry (ICP-MS) was performed toacquire data showing the selective uptake of drug by tumor tissue.

Tumor tissue was collected based on tumor location seen by MRI image. Asa normal tissue control, samples were collected from the same locationwithin the contralateral brain hemisphere. As a control tissue otherthan cerebral cortex, we also collected cerebellum to evaluate thesafety of our therapy.

FIG. 37 shows the results of ICP-MS performed 24 hours afteradministration to a rat of TLD1433 (5 mg/kg body weight) with humanApo-transferrin.

FIG. 38 shows the results of ICP-MS performed 4 hours afteradministration to a first rat of TLD1433 (5 mg/kg body weight) withOPTIFERRIN.

FIG. 39 shows the results of ICP-MS performed 24 hours afteradministration to a second rat of TLD1433 (5 mg/kg body weight) withOPTIFERRIN.

The data show that the PDC (TLD1433) is actively taken-up by tumortissue through the transferrin receptor. FIG. 37 shows that uptake ofthe PDC was 15.7 times higher in tumor tissue than in healthy tissuewhen it was injected as a mixture with human-Apo transferrin. Similarselective uptake was shown with Optiferrin (FIGS. 38 and 39).

Selectivity of PDT is provided by the pharmacokinetics of thephotosensitizers, leading to a local differential accumulation andretention in GBM tumor tissue versus normal intracranial tissues toprovide PDT specificity to tumor tissue. The data show that the PDC hasa good pharmacokinetic profile with a significantly higher concentrationof drug in tumor compared to normal tissue at 4 hrs and 24 hrs post druginjection. It is expected that this effect will persist over longerdurations.

Example 49 In Vitro PDT with TLD1433

A PRESTOBLUE method was performed to evaluate LD50 ofTLD1433+apo-transferrin on RG2 cells. On Day 1, cells were prepared in96-well plates in complete DMEM with phenol red: 15,000 RG2 cells/wellin 200 μl media. Two plates were prepared—one for dark toxicity and onefor 530 nm green 20 J PDT.

The plates were placed in a tissue culture incubator overnight.

On Day 2, drug dilutions of 0, 10, 25, 50, 75, 100, 200, 300, 400, 500were prepared. Media were removed from plates and washed with 100 μl ofmedia with no phenol red and sodium pyruvate. 100 μl of drug dilutionswere added to respective wells and incubated for 1 hour in tissueculture incubator. After 1 hour, the plates were washed once andrefilled with 100 μl fresh media. Then PDT was performed with a greenlaser head (#00346), 20 J/cm²=56 seconds. Later plates were returned toincubator for overnight.

On Day 3, PRESTOBLUE was prepared (1/10) in media without phenol red orsodium pyruvate. 100 μl PRESTOBLUE was added to each well and the plateswere read at 570/600 nm with 590 nm cut-off.

FIG. 40 is a graph showing LD50 calculations for TLD1433 mediated PDT ofRG2 cells. The LD50 was 47.34791+−9.06154 nM.

These results suggest that RG2 cells are highly sensitive to TLD1433mediated PDT.

Example 50 In Vitro PDT with TLD1433 and TLDOsH2IPdppn

Human glioblastoma (U87) cells were incubated in the presence ofruthenium-based (TLD1433) or osmium-based (TLDOsH2IPdppn, shown below):

photosensitizer without or with 0.4 mg/mL human apo-transferrin oroptiferrin for 2 hours at 37° C. If no transferrin or optiferrin wasadded, an equivalent volume of medium was added instead.

After that, the medium was replaced with a fresh one (withoutphotosensitizer and transferrin/optiferrin). For PDT, the cells wereirradiated (635 nm, 90 J/cm² for TLD1433 and 808 nm, 600 J/cm² forTLDOsH2IPdppn).

On the next day (21 hours post-irradiation), viability of the cells wasmeasured using a PRESTOBLUE viability assay, and the percent of cellkill was calculated. The PDT effect shown is a result of subtraction of“photosensitizer alone” and “light alone” cell kill from the total PDTcell kill.

As shown in FIGS. 41A-41D and 42A-42D, the presence oftransferrin/optiferrin decreases dark toxicity (photosensitizer alone)and increases the PDT effect of the photosensitizers.

This allows for safer PDT treatment in the presence oftransferrin/optiferrin due to shorter treatment time and the use oflower concentrations of the photosensitizers.

It is anticipated that similarly effective results will be achieved withrespect to other types of tumor cells, such as retinoblastoma cells.

While the invention has been described in detail and with reference tospecific examples thereof, it will be apparent to one skilled in the artthat various changes and modifications can be made therein withoutdeparting from the spirit and scope thereof.

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What is claimed is:
 1. A method for treating a condition associated withhyperproliferating cells, said method comprising administering to asubject having the condition a metal-binding glycoprotein and achemotherapeutic compound containing at least one transition metal,wherein the metal-binding glycoprotein and the chemotherapeutic compoundcontaining at least one transition metal are administered in a combinedamount effective to treat the condition.
 2. The method of claim 1,further comprising irradiating the subject with light effective toactivate the chemotherapeutic compound.
 3. The method of claim 2,wherein the metal-binding glycoprotein is effective to provide at leastone of the following advantages relative to treatment by thechemotherapeutic compound without the glycoprotein: (a) increased uptakeby cancer cells; (b) increased uptake by tumors; (c) increased efficacyat wavelengths longer than 600 nm; (d) increased efficacy at wavelengthsless than or equal to 600 nm; (e) improved absorbance at wavelengthslonger than 600 nm; (f) improved absorbance at wavelengths less than orequal to 600 nm; (g) increased production of reactive oxygen species;(h) increased photodynamic therapy effect under non-hypoxic conditions;(i) increased photodynamic therapy effect under hypoxic conditions; (j)increased LD50; (k) increased MTD; (l) increased photostability; and (m)increased shelf-life.
 4. The method of claim 1, wherein themetal-binding glycoprotein and the chemotherapeutic compound containingat least one transition metal are administered in a combination dosageform or are co-administered from two or more independent sources.
 5. Themethod of claim 1, wherein the metal-binding glycoprotein is transferrinand the chemotherapeutic compound has the formula (I):

including hydrates, solvates, pharmaceutically acceptable salts,prodrugs and complexes thereof, wherein: M at each occurrence isindependently selected from the group consisting of osmium, rutheniumand rhodium; X is selected from the group consisting of Cl⁻, PF₆ ⁻, Br⁻,BF₄ ⁻, ClO₄ ⁻, CF₃SO₃ ⁻, and SO₄ ⁻²; n=0, 1, 2, 3, 4, or 5; q isindependently at each occurrence 0, 1, or 2; y is independently at eachoccurrence 0, 1, or 2; z is independently at each occurrence 1, 2, or 3;Lig¹ is a bidentate ligand that at each occurrence is each independentlyselected from the group consisting of

Lig² is a bidentate ligand that at each occurrence is each independentlyselected from the group consisting of

Lig³ is a bidentate ligand that at each occurrence is each independentlyselected from the group consisting of

R¹ is selected from the group consisting of hydrogen, optionallysubstituted phenyl, optionally substituted aryl, optionally substitutedheteroaryl, 4-pyridyl, 3-pyridyl, 2-thiazole, 2-pyrolyl, 2-furanyl,

u is an integer; R^(2a), R^(2b), R^(2c), R^(2d), R^(2e), R^(2f), R^(2g),R^(2h), R^(2i), R^(2j), R^(2k), and R^(2l) at each occurrence are eachindependently selected from the group consisting of hydrogen, C₁₋₆optionally substituted alkyl, C₁₋₆ optionally substituted branchedalkyl, C₃₋₇ optionally substituted cycloalkyl, C₁₋₆ optionallysubstituted haloalkyl, C₁₋₆ optionally substituted alkoxy, CO₂R⁵, CONR⁶₂, NR⁷ ₂, SO₃H, sulfate, sulfonate, optionally substituted aryl,optionally substituted aryloxy, optionally substituted heteroaryl, andoptionally substituted heterocycle; R^(3a), R^(3b), R^(3c), R^(3d),R^(3e), R^(3f), R^(3g), R^(3h) R^(3i), R^(3j), R^(3k), and R^(3l) ateach occurrence are each independently selected from the groupconsisting of hydrogen, C₁₋₆ optionally substituted alkyl, C₁₋₆optionally substituted branched alkyl, C₁₋₆ optionally substitutedhaloalkyl, C₁₋₆ optionally substituted alkoxy, optionally substitutedphenyl, and CO₂R⁸; R^(4a), R^(4b), and R^(4c) at each occurrence areeach independently selected from the group consisting of hydrogen, C₁₋₆optionally substituted alkyl, C₁₋₆ optionally substituted branchedalkyl, C₁₋₆ optionally substituted cycloalkyl, C₁₋₆ optionallysubstituted haloalkyl, C₁₋₆ optionally substituted alkoxy, CO₂R⁵, CONR⁶₂, NR⁷ ₂, sulfate, sulfonate, optionally substituted aryl, optionallysubstituted aryloxy, optionally substituted heteroaryl, and optionallysubstituted heterocycle; R^(4a) and R^(4b) at each occurrence on athiophene ring are taken together with the atom to which they are boundto form an optionally substituted ring having from 6 ring atomscontaining 2 oxygen atoms; R⁵ at each occurrence are each independentlyselected from the group consisting of hydrogen and optionallysubstituted alkyl; R⁶ at each occurrence are each independently selectedfrom the group consisting of hydrogen and optionally substituted alkyl;R⁷ at each occurrence are each independently selected from the groupconsisting of hydrogen and optionally substituted alkyl; and R⁸ at eachoccurrence are each independently selected from the group consisting ofhydrogen and optionally substituted alkyl.
 6. The method of claim 1,wherein the metal-binding glycoprotein is transferrin and thechemotherapeutic compound has the formula (VI):

including hydrates, solvates, pharmaceutically acceptable salts,prodrugs and complexes thereof wherein; M¹ and M² at each occurrence isindependently selected from the group consisting of osmium, manganese,molybdenum, rhenium, ruthenium, iron, cobalt, rhodium, iridium, nickel,platinum, and copper; A² is selected from the group consisting of

t is an integer.
 7. The method of claim 1, wherein the metal-bindingglycoprotein is transferrin and the chemotherapeutic compound has theformula (VIIa)

including hydrates, solvates, pharmaceutically acceptable salts,prodrugs and complexes thereof wherein: A³ is selected from the groupconsisting of

Lig¹ is a bidentate ligand that at each occurrence is each independentlyselected from the group consisting of

Lig³ is a bidentate ligand that at each occurrence is each independentlyselected from the group consisting of

R¹ is selected from the group consisting of hydrogen, optionallysubstituted phenyl, optionally substituted aryl, optionally substitutedheteroaryl, 4-pyridyl, 3-pyridyl, 2-thiazole, 2-pyrolyl, 2-furanyl,

u is an integer; R^(2a), R^(2b), R^(2c), R^(2d), R^(2e), R^(2f), R^(2g),R^(2h), R^(2i), R^(2j), R^(2k), and R^(2l) at each occurrence are eachindependently selected from the group consisting of hydrogen, C₁₋₆optionally substituted alkyl, C₁₋₆ optionally substituted branchedalkyl, C₃₋₇ optionally substituted cycloalkyl, C₁₋₆ optionallysubstituted haloalkyl, C₁₋₆ optionally substituted alkoxy, CO₂R⁵, CONR⁶₂, NR⁷ ₂, SO₃H, sulfate, sulfonate, optionally substituted aryl,optionally substituted aryloxy, optionally substituted heteroaryl, andoptionally substituted heterocycle; R^(3a), R^(3b), R^(3c), R^(3d),R^(3e), R^(3f), R^(3g), R^(3h) R^(3i), R^(3j), R^(3k), and R^(3l) ateach occurrence are each independently selected from the groupconsisting of hydrogen, C₁₋₆ optionally substituted alkyl, C₁₋₆optionally substituted branched alkyl, C₁₋₆ optionally substitutedhaloalkyl, C₁₋₆ optionally substituted alkoxy, optionally substitutedphenyl, and CO₂R⁸; R^(4a), R^(4b), and R^(4c) at each occurrence areeach independently selected from the group consisting of hydrogen, C₁₋₆optionally substituted alkyl, C₁₋₆ optionally substituted branchedalkyl, C₁₋₆ optionally substituted cycloalkyl, C₁₋₆ optionallysubstituted haloalkyl, C₁₋₆ optionally substituted alkoxy, CO₂R⁵, CONR⁶₂, NR⁷ ₂, sulfate, sulfonate, optionally substituted aryl, optionallysubstituted aryloxy, optionally substituted heteroaryl, and optionallysubstituted heterocycle; R^(4a) and R^(4b) at each occurrence on athiophene ring are taken together with the atom to which they are boundto form an optionally substituted ring having from 6 ring atomscontaining 2 oxygen atoms; R⁵ at each occurrence are each independentlyselected from the group consisting of hydrogen and optionallysubstituted alkyl; R⁶ at each occurrence are each independently selectedfrom the group consisting of hydrogen and optionally substituted alkyl;R⁷ at each occurrence are each independently selected from the groupconsisting of hydrogen and optionally substituted alkyl; and R⁸ at eachoccurrence are each independently selected from the group consisting ofhydrogen and optionally substituted alkyl p is independently at eachoccurrence 0, 1, or 2; q is independently at each occurrence 0, 1, or 2;and n is 0, 1, 2, 3, 4, or
 5. 8. The method of claim 1, wherein themetal-binding glycoprotein is transferrin and the chemotherapeuticcompound has the formula (II)

including hydrates, solvates, pharmaceutically acceptable salts,prodrugs and complexes thereof, wherein: M is selected from the groupconsisting of manganese, molybdenum, rhenium, iron, ruthenium, osmium,cobalt, rhodium, iridium, nickel, platinum, and copper; X is selectedfrom the group consisting of Cl⁻, PF₆ ⁻, Br⁻, BF₄ ⁻, ClO₄ ⁻, CF₃SO₃ ⁻,and SO₄ ⁻²; n=0, 1, 2, 3, 4, or 5; y=1, 2, or 3; z=0, 1, or 2; Lig ateach occurrence is independently selected from the group consisting of

R¹ is selected from the group consisting of

u is an integer; R^(2a), R^(2b), R^(2c), R^(2d), R^(2e), and R^(2f) ateach occurrence are each independently selected from the groupconsisting of hydrogen, C1-6 optionally substituted alkyl, C1-6optionally substituted branched alkyl, C3-7 optionally substitutedcycloalkyl, C1-6 optionally substituted haloalkyl, C1-6 optionallysubstituted alkoxy, CO₂R⁵, CONR⁶ ₂, NR⁷ ₂, sulfate, sulfonate,optionally substituted aryl, optionally substituted aryloxy, optionallysubstituted heteroaryl, and optionally substituted heterocycle; R^(3a),R^(3b), R^(3c), R^(3d), R^(3e), R^(3f), R^(3g), R^(3h) R^(3i), R^(3j),R^(3k), R^(3l), and R^(3m) at each occurrence are each independentlyselected from the group consisting of hydrogen, C1-6 optionallysubstituted alkyl, C1-6 optionally substituted branched alkyl, C1-6optionally substituted haloalkyl, C1-6 optionally substituted alkoxy,and CO₂R⁸; R^(4a), R^(4b), and R^(4c) at each occurrence are eachindependently selected from the group consisting of hydrogen, C1-6optionally substituted alkyl, C1-6 optionally substituted branchedalkyl, C1-6 optionally substituted cycloalkyl, C1-6 optionallysubstituted haloalkyl, C1-6 optionally substituted alkoxy, CO₂R⁵, CONR⁶₂, NR⁷ ₂, sulfate, sulfonate, optionally substituted aryl, optionallysubstituted aryloxy, optionally substituted heteroaryl, and optionallysubstituted heterocycle; R^(4a) and R^(4b) at each occurrence on athiophene ring are taken together with the atom to which they are boundto form an optionally substituted ring having from 6 ring atomscontaining 2 oxygen atoms; R⁵ at each occurrence is independentlyselected from the group consisting of hydrogen and optionallysubstituted alkyl; R⁶ at each occurrence is independently selected fromthe group consisting of hydrogen and optionally substituted alkyl; R⁷ ateach occurrence is independently selected from the group consisting ofhydrogen and optionally substituted alkyl; and R⁸ at each occurrence isindependently selected from the group consisting of hydrogen andoptionally substituted alkyl.
 9. The method of claim 1, wherein the atleast one transition metal is at least one of Ru, Rh, Os and Ir.
 10. Themethod of claim 1, wherein the metal-binding glycoprotein is arecombinant human transferrin.
 11. The method of claim 1, wherein thecondition treated by the method is at an immune privileged site, themetal-binding glycoprotein and the chemotherapeutic compound containingat least one transition metal are systemically administered in a form ofa complex, and the complex crosses at least one of the blood-brainbarrier, the retina-blood barrier and the blood-cerebrospinal fluidbarrier to accumulate at the immune privileged site.
 12. The method ofclaim 11, wherein the complex crosses the blood-brain barrier andpreferentially accumulates in a brain tumor being treated by the method.13. The method of claim 3, wherein the condition treated by the methodis at an immune privileged site, the metal-binding glycoprotein and thechemotherapeutic compound containing at least one transition metal aresystemically administered in a form of a complex, and the complexcrosses at least one of the blood-brain barrier, the retina-bloodbarrier and the blood-cerebrospinal fluid barrier to accumulate at theimmune privileged site.
 14. The method of claim 5, wherein the conditiontreated by the method is at an immune privileged site, the metal-bindingglycoprotein and the chemotherapeutic compound containing at least onetransition metal are systemically administered in a form of a complex,and the complex crosses at least one of the blood-brain barrier, theretina-blood barrier and the blood-cerebrospinal fluid barrier toaccumulate at the immune privileged site.
 15. The method of claim 6,wherein the condition treated by the method is at an immune privilegedsite, the metal-binding glycoprotein and the chemotherapeutic compoundcontaining at least one transition metal are systemically administeredin a form of a complex, and the complex crosses at least one of theblood-brain barrier, the retina-blood barrier and theblood-cerebrospinal fluid barrier to accumulate at the immune privilegedsite.
 16. The method of claim 7, wherein the condition treated by themethod is at an immune privileged site, the metal-binding glycoproteinand the chemotherapeutic compound containing at least one transitionmetal are systemically administered in a form of a complex, and thecomplex crosses at least one of the blood-brain barrier, theretina-blood barrier and the blood-cerebrospinal fluid barrier toaccumulate at the immune privileged site.
 17. The method of claim 8,wherein the condition treated by the method is at an immune privilegedsite, the metal-binding glycoprotein and the chemotherapeutic compoundcontaining at least one transition metal are systemically administeredin a form of a complex, and the complex crosses at least one of theblood-brain barrier, the retina-blood barrier and theblood-cerebrospinal fluid barrier to accumulate at the immune privilegedsite.
 18. The method of claim 1, further comprising irradiating thesubject with ionizing radiation effective to activate thechemotherapeutic compound.
 19. The method of claim 1, wherein themetal-binding glycoprotein and the chemotherapeutic compound containingat least one transition metal are administered systemically in a form ofa complex, the chemotherapeutic compound is a photodynamic compound, andthe complex further comprises at least one active pharmaceuticalingredient.
 20. The method of claim 19, wherein the at least one activepharmaceutical ingredient is an anti-neoplastic agent that is free ofany transition metals.
 21. The method of claim 19, wherein the complexcrosses at least one of the blood-brain barrier, the retina-bloodbarrier and the blood-cerebrospinal fluid barrier to accumulate at animmune privileged site.